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Bioavailable Iron and Vitamin A in Newly Formulated, Extruded Corn, Soybean, Sorghum and Cowpea Fortified-Blended Foods in the In-vitro Digestion/Caco-2 Cell Model

Bioavailable Iron and Vitamin A in Newly Formulated, Extruded Corn, Soybean, Sorghum and Cowpea... Bioavailable Iron and Vitamin A in Newly Formulated, Extruded Corn, Soybean, Sorghum and Cowpea Fortified-Blended Foods in the In-vitro Digestion/Caco-2 Cell Model 1 1 2, 1 Kavitha Penugonda , Nicole Fiorentino , Sajid Alavi and Brian L. Lindshield * Department of Food, Nutrition, Dietetics and Health. Kansas State University, Manhattan, KS 66506, USA Department of Grain Science and Industry, Manhattan, KS 66506, USA * Corresponding author: Brian Lindshield e-mail: blindsh@k-state.edu Tel.: +1-785-532-7848 Fax: +1-785-532-3132. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Abbreviations used: BHT, butylated hydroxytoluene; CSB, corn-soy blend; CSB+, corn-soy blend plus; FAQR, food aid quality review; FBFs, Fortified blended foods; FeSO , ferrous sulfate; KOH, potassium hydroxide; MFFAPP, Micronutrient Fortified Food Aid Products Pilot Program; NaFeEDTA, sodiu m iron EDTA; SCB, sorghum-cowpea blend; SSB, sorghum-soy blend; USAID, United States Agency for International Development; USDA, United States Department of Agriculture; WPC 80, whey protein concentrate with 80% protein content; Sources of financial support: This study was supported by the United States Department of Agriculture Micronutrient Fortified Food Aid Products Pilot Program (MFFAPP), contract number #FFE-621-2012/033-00. This is contribution no. 17-019-J of the Kansas Agricultural Experiment Station, Manhattan, KS. Conflict of Interest: The authors declare no conflicts of interest. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Abstract Background: Fortified blended foods (FBFs), particularly corn-soybean blend (CSB), are food aid products distributed in developing countries. The United States Agency for International Development (USAID) food aid quality review recommended developing extruded FBFs using alternative commodities such as sorghum. Objective: The objective of the study was to determine bioavailable iron and vitamin A levels, from newly developed, extruded corn, soybean, sorghum, and cowpea FBFs compared to the non-extruded traditional food aid FBFs, corn-soy blend 13 and corn-soy blend plus (CSB+). Methods: Eleven extruded FBFs; sorghum-cowpea (n=7), sorghum-soy (n=3), and corn-soy (n=1), along with two non-extruded FBFs; corn-soy blend 13 (CSB13) and corn-soy blend plus (CSB+), and Cerelac, a commercially available fortified infant food, were prepared. Bioavailable iron and vitamin A levels were assessed using the in-vitro digestion/Caco-2 cell model. Dry FBFs, aqueous fractions, and Caco-2 cell pellets vitamin A levels were analyzed by HPLC. Dry FBFs and aqueous fraction iron levels were measured by atomic absorptiometry and bioavailab le iron assessed by measuring Caco-2 ferritin levels via ELISA. Results: Iron and vitamin A content in Cerelac and dry FBFs ranged from 8.0 to 31.8 mg/100g and 0.3 to 1.67 mg/100g, respectively. All of the extruded FBFs contained 4- to 7-fold significantly higher (p<0.05) aqueous fraction iron concentrations compared to CSB13 and CSB+. However, Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 there were no significant differences in Caco-2 cell ferritin and vitamin A levels between extruded FBFs, non-extruded FBFs, or the basal salt solution negative control. Conclusions: Results support that consumption of newly developed extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs would result in iron and vitamin A levels comparable to traditional non-extruded CSB13 and CSB+ FBFs. Keywords: Fortified blended food; corn-soy blend plus; micronutrient bioavailability; iron; vitamin A, whey protein concent rate, FAQR; food aid; Title II foods, in-vitro digestion/Caco-2 cell model. Introduction Fortified blended foods (FBFs) are porridge mixes composed of cereals and legumes that have been milled and fortified with vitamins and minerals. FBFs are major food aid products for young children, women, and other vulnerable groups in developing countries. Historically corn-soy blend (CSB) has been the most widely distributed FBF in a majority of the food aid receiving countries [1]. The United States Agency for International Development (USAID) Food Aid Quality Review (FAQR) recommended developing novel FBFs using cereals that are both culturally and nutritionally acceptable in Africa. It also recommended sorghum as an alternative to corn or wheat and suggests other legumes could be paired with it as alternatives to soy [2]. One logical legume to investigate is cowpea because Africa is the world leading producer of cowpea (95%) in addition to sorghum (41%) [3]. Both sorghum and cowpea are drought-tolerant, sustainable, and not genetically modified (Non-GMO) grains, which is preferred by some food aid recipient nations. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Sorghum and cowpea are rich in iron and are complementary proteins [4, 5], however, sorghum and cowpea also contain the antinutritional factors, phytates, and tannins, which impair iron bioavailability [6-9]. Extrusion is a food processing technique that cooks food using high temperature under high pressure in combination with moisture and mechanical shear [10]. The desirable effects of this cost-effective method are that it decreases viscosity; increases palatability, starch and protein digestibility; and reduces anti-nutritional factor levels thereby potentially improving iron bioavailability [11, 12]. Extruded novel sorghum-cowpea, sorghum-soy, and corn-soy FBFs were developed based on the USAID FAQR recommendations [2] and USDA commodity requirements [13, 14] as part of the Micronutrient Fortified Food Aid Pilot Project [15]. Traditional, non-extruded FBFs, CSB13, and CSB+, were procured to use as comparisons for the newly developed FBFs. The purpose of this study was to assess bioavailable iron and vitamin A levels of newly developed extruded sorghum-cowpea blend (SCB), sorghum-soy blend (SSB), and corn-soy blend (CSB14) FBFs compared to traditional non-extruded FBFs, CSB13 and CSB+, in the in- vitro digestion/Caco-2 cell model. These micronutrients deficiencies were chosen because they are a substantial public health issue for many women and children throughout the world [16]. The in-vitro digestion/Caco-2 model was used because it is a widely used, inexpensive model to study the bioavailability of nutrients from foods and supplements [17-21]. It has been successfully used to screen for iron bioavailability of a variety of complementary foods [22], lentils [23], wheat [24], cassava [25], and supplemented food stuffs [26]. We use the term “bioavailable” to describe the amount of compound in the Caco-2 cells after they were treated with aqueous fractions produced by in-vitro digestion. To the best of our knowledge, this is the first study to use this model to assess both bioavailable iron and vitamin A. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Materials and Methods Chemicals Unless stated otherwise, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). Double deionized water was used for porridge preparation, in-vitro digestion, reagent preparation, and vitamin A extraction. To prevent iron contamination, glassware used in the sample preparation, in-vitro digestion, and iron analysis were acid washed by soaking in 5% nitric acid solution for no less than 12 hours and rinsing with double deionized water before use. Acetonitrile, methanol, chloroform, hexane, and ethanol were HPLC grade. FBF Formulations Extruded sorghum-cowpea (n=7), sorghum-soy (n=3), and corn-soy (n=1) were formulated based on FAQR requirements [2]. Two white (variety 1, Fontanelle 4575; variety 2, 738Y) and one red (217X Burgundy) sorghum varieties, whole or decorticated, were used in producing extruded sorghum-cowpea FBFs and cowpea flour was sourced commercially (Table 1 & Table 2).Extruded sorghum-soy FBFs contained white sorghum variety 1 (Fontanelle 4575), whole or decorticated, with low-fat (1.85%), medium-fat (6.94%), or full-fat (16.93%) soy. Extruded CSB14 was formulated with degermed corn with medium-fat soy. The other FBF components; sugar, oil, whey protein concentrate with 80% protein content (WPC 80), and vitamin-mineral premix were added after extrusion to prevent destruction of micronutrients. Both non-extruded FBFs, CSB13 and CSB+, were purchased from Bunge Milling (St. Louis, MO). The major difference between these two CSBs is that CSB+ is a more recently released CSB with heat-processed corn and soy and improved micronutrient formulation [27]. Cerelac (Nestle, NJ), a commercially available fortified infant food, was purchased from a local store and included as a reference control as has been done previously Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 [22]. It is worth noting that the iron and vitamin A fortificants differed between Cerelac, extruded, and non-extruded FBFs. Extruded FBFs contained ferrous sulfate (FeSO ) and sodium iron EDTA (NaFeEDTA); CSB+ contained ferrous fumarate and NaFeEDTA; CSB13 and Cerelac contained only ferrous fumarate (Table 3). Extruded and non-extruded FBFs contained retinyl palmitate, while Cerelac contained retinyl acetate. All the FBFs were stored at -20°C in zip lock bags covered with aluminum foil. FBF Porridge Preparation Twenty grams dry FBF or Cerelac was slowly added to 80 g of boiling water in a beaker on a hot plate and stirred vigorously for 2 min, removed from hot plate and stirred for another minute. Non-extruded CSB13 and CSB+ were prepared in a similar manner but 11.75 g and 13.79 g dry FBF, respectively, was used and they were cooked for 10 min on a hot plate following the preparation instructions for CSB+ [13, 14]. Porridges were then covered with aluminum foil and kept in water bath at 37°C for 10 min to prevent skin formation. Porridges were then weighed and water lo st during preparation was added back in to bring the final weight to 100 g. Porridges were transferred to 50 ml polypropylene tubes, co vered in aluminum foil, blanketed with nitrogen, sealed and stored at -80°C. Porridges were prepared in duplicate on two different days (4 replicates). Later, these replicates were used for in-vitro digestion/Caco-2 cell experiments. In-vitro Digestion Porridge aliquots were subjected to in-vitro digestion as described previously [28]. Ten ml of basal salt solution (120 mmol/L NaCl, 5 mmol/L KCl, and 6 mmol/L CaCl ) was added to 2.5 grams of thawed FBF in a beaker, homogenized with a laboratory homogenizer for 2 min, and then Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 mixed on a magnetic stir plate for 5 min. Ten ml aliquots of homogenized FBFs were then subjected to three continuous in-vitro digestion phases: 10 min oral, 1-hour gastric, and 2-hour small intestine. Oral Digestion Saliva solution containing 0.9 mg KCl, 0.89 mg NaPO , 0.57 mg NaSO , 0.3 mg NaCl, and 1.69 mg NaHCO /ml deionized water was prepared 4 4 3 and used for all experiments. Ten ml of homogenized FBF solution and 8 ml of freshly prepared artificial saliva [(uric acid (0.015 mg/ml), urea (0.2 mg/ml), mucin (0.025 mg/ml), and α–amylase (10.55 mg/ml) dissolved in saliva solution)] were added to a 50 ml conical tube. Tubes were mixed well, blanketed with nitrogen, sealed with parafilm and incubated placing horizontally in a shaking water bath at 37°C, 85 rp m for 10 min in the dark. Gastric Digestion After oral digestion, digesta pH was decreased to 2.5 ± 0.1 by slowly adding 1M HCl and then 2 ml of freshly made pepsin solution (40 mg/ml in 100mM HCl) was added. The final volume was then adjusted to 40 ml with basal salt solution, blanketed with nitrogen, sealed with parafilm and incubated in a shaking water bath at 37°C, 85 rpm for 1 hour in the dark. Small Intestinal Digestion The gastric phase was terminated by increasing digesta pH to 6.0 ± 0.1 with 1M NaHCO and placing the tubes on ice. Two ml of pancreatin (10 mg/ml) and lipase (5 mg/ml) solution (both in in 100mM NaHCO ) was then added along with 3 ml of bile extract (40 mg/ml 100mM NaHCO ) and the digesta pH was 3 3 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 adjusted to 6.5 ± 0.1 with 1 M NaOH. The final volume was adjusted to 50 ml with basal salt solution, blanketed with nitrogen, sealed with parafilm and incubated in a shaking water bath at 37°C, 85 rpm for 2 hours in the dark. Isolation of Aqueous Fraction from Digesta After small intestine digestion, 10 ml digesta aliquots were transferred to 15 ml polypropylene tubes and centrifuged at 5000 g for 45 min at 5°C. Supernatants were collected by puncturing the side of the tube with an 18 gauge needle and 10 ml syringe without disturbing the pellet. Supernatants were filtered using 0.22 µm syringe filters (SLGP 033 RS, Millipore, MA), and fresh aqueous fractions were used to treat the Caco-2 cells. Aqueous fractions aliquots were blanketed with nitrogen and stored at -80°C for later analysis [28]. Dry Porridge Blends and Aqueous Fractions Iron Determination Dry porridge blend and aqueous fraction iron content were analyzed using an atomic absorption spectrophotometer (AAS). Dry po rridge blends’ iron levels were analyzed (AACC 40-70, 1999) by American Institute of Baking International Analytical Services (Manhattan, KS). Briefly, ten grams of sample was taken in ashing vessels and dried to ash overnight at 500°C in a muffle furnace. Residue was dissolved in 10 ml of concentrated HCl, boiled and evaporated to near dryness on a hot plate. Resulting residue was redissolved in 20 ml of 2N HCl, filtered and diluted to 100 ml with water. Iron concentrations were then measured on an atomic absorption spectrophotometer. Aqueous fractions’ iron concentratio ns, filtered samples, were directly measured on an AAS (Perkin Elmer, AAnalyst 100). Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Caco-2 Cell Cultures Caco-2 cells (ATCC® HTB37™) purchased from American Type Culture Collection (Manassas, VA) were used in the experiment at passage 32 and 33. The cells were maintained at 37°C in an incubator with a 5% CO /95% humidity, and media was changed every other day. Caco-2 cells were initially cultured in growth-enhanced treated T-75 flasks (TP 90076, Midsci, MO) in the presence of Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Grand Island, NY), supplemented with 15% fetal bovine serum (FBS, Atlanta Biologicals, GA), 1% L-glutamine, 1% non-essential amino acids, 1% antibiotic/antimycotic (penicillin/streptomycin) solution, and 0.2% amphotericin B [28]. Confluent cells were subcultured by incubating with 5 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) solution for 5 min, which was then inactivated by adding 10 ml of 15% DMEM. After trypsinization, cell suspensions were collected into 50 ml conical tubes, centrifuged at 800 rpm for 5 min at room temperature, the supernatant media discarded and the cell pellet collected. After re-suspending the cell pellet and counting with a hemocytometer, cells were seeded at 50,000 cells/cm in tissue culture treated 6-well plates (Corning Inc, Corning, NY). After being seeded at day 0, the cells usually became confluent 4-5 days later, at which point they were switched from media containing 15% FBS to 7.5% FBS to slow growth. Cells were used in the iron and vitamin A bioavailability experiments 14 days post-seeding [29, 30]. Aqueous Fraction Caco-2 Treatment On day 13, a day before the experiment, Caco-2 monolayers were provided fresh media. On day 14, media was removed before treating the cells with 0.25 ml fresh aqueous fraction and 1.75 ml of DMEM for iron or 0.5 ml fresh aqueous fraction and 1.5 ml of DMEM for vitamin A, which were then incubated for 12 [31, 32] or 4 hours [33, 26, 25], respectively. Samples were randomly assigned to wells; Cerelac was used as a reference Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 control on each plate. A negative control was prepared with 0.25 ml of basal salt solution containing no iron and 1.75 ml of DMEM. A positive control was prepared with basal salt solution to provide 0.1 µg iron/well or 0.2 µg iron/well. These iron concentrations were selected to match with the estimated iron concentration in the digested FBF aliquots that were added to the Caco-2 cells. In-vitro digestion and Caco-2 cell culture experiments were completed in duplicate on different days using different cells passages. Caco-2 Ferritin and Protein Determination After incubation, treatments were removed and cells were washed with 2 ml of ice cold 2X PBS. Caco -2 monolayers were lysed by adding 350 µl/well of mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific, Rockford, IL) [34] and incubated in 6-well plates for 10 minutes on a plate shaker at 120 rpm. Caco-2 monolayers were scraped with a cell scraper (Fisher Scientific, Pittsburgh, PA), collected into microcentrifuge tubes, sonicated for 3 minutes and centrifuged at 14,000g for 10 minutes. Cell lysate supernatants were transferred to microcentrifuge tubes and stored at -20°C for ferritin and protein determination, which was completed within 24 hours [34, 35, 21]. Ten µl cell lysate solutions were used for determining ferritin concentrations (ng/ml) using enzyme-linked immunosorbent assay (ELISA, Spectro Ferritin kit, S- 22, Ramco Laboratories Inc., Stafford, TX) as done previously [23, 35]. Twenty five µl cell lysate solutions were used for measuring protein concentrations using Pierce bicinchoninic acid protein assay kits (Rockford, IL). Ferritin content (ng/mg cell protein) was c alculated as a ratio of cell ferritin (ng/ml)/cell protein (mg/ml) [35]. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Harvesting Caco-2 Monolayers for Vitamin A Assessment After incubation, treatments were removed and cells were washed with 2 ml of ice cold 2X PBS followed by 2 ml of ice cold 2 g /L albumin in PBS. After discarding the washing solutions, Caco-2 monolayers were removed using a cell scraper (Fisher Scientific, Pittsburgh, PA), and collected into amber colored microcentrifuge tubes using 1 ml ice cold PBS. This process was repeated twice more using 0.5 ml ice cold PBS, for a total of 2 ml PBS collected into the same microcentrifuge tubes and centrifuged at 2000 rpm for 45 minutes at 5°C. Supernatant PBS was discarded by carefully inverting the microcentrifuge tubes for 20 seconds. Tubes with cell pellets were then blanketed with nitrogen, and stored at -80°C for vitamin A analysis [33]. Extraction of Vitamin A in Dry Porridge, Aqueous Fraction and Caco-2 Cells Vitamin A was extracted from dry porridge, aqueous fractions and Caco-2 cell pellets as described previously [36] with modifications. For dry porridge, approximately 1 g of dry porridge in a 50 ml screw cap glass tube was homogenized in 4 ml of deionized water before adding 10 ml of ethanol with 0.1% butylated hydroxytoluene (BHT) and 4 mL of super saturated potassium hydroxide (KOH) solution. Samples were vortexed and incubated in a water bath at 70°C for 30 minutes, vortexing every 10 minutes. The tubes were placed on ice and 6 ml of deionized distilled water was added. The samples were initially extracted with 10 ml of hexane and then twice with 5 ml of hexane. Tubes were vortexed each time hexane was added and left on ice to allow layer separation. The top hexane layer was collected into a clean glass tube with Pasteur pipette, completely dried in vacufuge (Model No, 5301, Eppendorf North America, Hauppauge, NY, USA), and reconstituted in 400 µl of mobile phase. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 For aqueous fractions, 8 ml of the thawed aqueous fraction was extracted with 10 ml of ethanol with 0.1% BHT and 4 mL of supe r saturated KOH. The rest of the extraction procedure was the same as described for dry porridge. Finally, reconstituted (in 400 µl mobile phase) aqueous fractions were syringe filtered using Whatman PVDF filters with 0.45µm pore size (Cat No. 6779-1304, GE Healthcare Biosciences, Pittsburgh, PA, USA) before placing in HPLC vials. Cell pellets were weighed then 2 ml of ethanol with 0.1% BHT and 1 mL of super saturated KOH were added and incubated at 70°C for 30 minutes, vortexing every 10 minutes [26]. The tubes were kept on ice and 1 ml of deionized distilled water was added. Samples were extracted with 5 ml of hexane three times and the hexane layers were dried in the manner similar to dry porridges. Dried hexane layers were reconstituted in 400 µl of mobile phase. HPLC Analysis All samples were analyzed the same day they were extracted. A Shimadzu HPLC system (Kyoto, Japan) consisting of a DGU-20A3 built in degasser, a LC-20AB solvent delivery pump, a SIL-20ACHT auto-sampler, a CTO-20AC column holding oven, a CBM-20A communicator module, and a SPD-M20A Photodiode Array Detector with an Agilent Eclipse XDB 5mm C18 (250mm x 4.6mm, Santa Clara, CA, USA) analytical column 25°C was used for analysis. A mobile phase of methanol/acetonitrile/chloroform (47:47:6, v/v/v) at a flow rate of 1.0 mL/min was used, detection was at 325 nm [37] and data was analyzed using LC solution software (Kyoto, Japan). An external retinyl acetate (catalog # 1716002, USP, Rockville, MD, USA) standard curve was used for quantification, with its concentration determined using a spectrophotometer ( Jenway 6305, Bibby Scientific US, NJ, USA). Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Statistical Analysis Data were analyzed using one-way ANOVA with Tukey’s test on SAS 9.3 (SAS Institute Inc., Cary, NC, USA), with p < 0.05 considered significant. Natural logs were used to transform data that did not meet the model assumptions. Results Iron Concentration in Dry FBF and Aqueous Fraction Dry FBFs’ iron concentration ranged from 8.0 to 31.8 mg/100 g (Table 3). Extruded dry FBF iron concentrations (15.1 to 23.4 mg/100 g) were higher than CSB+ (8.0 mg/100 g), but lower than CSB13 (31.8 mg/100 g). Cerelac’s iron concentration (11.0 mg/100 g) was lower than all the FBFs except CSB+. Among the sorghum-containing FBFs, iron levels were higher in whole sorghum compared to the decorticated FBFs. Aqueous fractions iron levels ranged from 0.14 to 1.03 µg/ml (Table 3). All extruded FBFs had significantly higher aqueous fractions iron levels compared to CSB13 and CSB+. There were no significant differences between extruded FBFs or between CSB13, CSB+, and Cerelac aqueous fraction levels. Cerelac aqueous fraction iron levels (0.42 µg/ml) were significantly lower compared to whole white sorghum1 -cowpea, decorticated white sorghum1-cowpea, whole sorghum-soy (low-fat), and whole sorghum-soy (full-fat) extruded FBFs. Ferritin Concentration in Caco-2 Cells Treated With Aqueous Fractions There were no significant differences between FBF aqueous fraction and basal salt solution (negative control) treatment ferritin levels (Table 5). Among the extruded FBFs, CSB14 had the highest ferritin levels (6.78 ng/mg) and whole sorghum-soy (full-fat) had the lowest ferritin levels (4.10 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 ng/mg). The FeSO positive control (0.2 µg iron/well) treatment resulted in significantly higher ferritin levels than Cerelac, negative co ntrol and all FBFs except for corn-soy (medium-fat) and CSB+. There was a dose-dependent increase in ferritin levels in response to FeSO treatment. Vitamin A Concentration in Dry FBF and Aqueous Fraction Dry FBFs’ vitamin A concentrations ranged from 0.54-1.67 mg/100 g (Table 6). CSB+ (1.67 mg/100 g) and Cerelac (0.3 mg vitamin A/100 g) had the highest and lowest vitamin A concentrations, respectively. Whole sorghum FBFs had slightly higher vitamin A levels co mpared to their corresponding decorticated sorghum FBFs. FBFs’ and Cerelac’s aqueous fraction vitamin A concentrations were similar and not significantly different. However, in general, sorghum-cowpea FBFs contained higher levels (50.8-80.1 ng/ml) than sorghum-soy and corn-soy FBFs (33.0-49.1 ng/ml). Vitamin A Concentration in Caco-2 Cells Treated With Aqueous Fraction Caco-2 cell pellet vitamin A concentrations were not significantly different, following aqueous fraction treatment, between extruded, non-extruded FBFs and Cerelac (Table 7). The interesting trend was that red sorghum levels were lower than white sorghum. Sorghum-soy FBFs with full-fat and medium-fat soy had nonsignificantly higher vitamin A levels than low-fat sorghum-soy FBF. The vitamin A levels in all the FBFs and Cerelac are higher than the negative control levels of 2.09 µg/g, with the exception of red sorghum FBFs, white whole sorghum-soy (low-fat soy), CSB13, and CSB14. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Discussion Seven of the extruded FBFs and one non-extruded FBF had ferritin response higher than negative control levels, but none were significantly higher (Table 5). It should be noted that previous studies conducted with similar food matrix have also not observed increases above negative control ferritin response. In a study comparing standard and iron biofortified black beans using the in vitro digestion/Caco-2 model, ferritin response for black bean varieties was significantly lower than the negative control [39]. In another study comparing red and white beans using the in vitro digestion/Caco-2 model, all sample ferritin responses were at, or significantly below, the negative control [38]. It should also be noted that the same manuscript found that white bean consumption resulted significant improvements in iron status compared to red bean consumptio n in pigs [38]. Rats fed dry extruded sorghum-cowpea, extruded sorghum-soy and extruded corn-soy (CSB14) FBFs or CSB+ showed no significant difference in hemoglobin and liver iron levels consistent with our Caco-2 results reported here [40]. The amount of iron added to Caco-2 cells in each well ranged from 0.11-0.25 µg/well, which was similar to the range (0.05- 0.39 µg/well) found in different rice varieties that also did not significantly increase ferritin levels [20]. However, adding ascorbic acid, iron bioavailability of the rice sample significantly increased beyond the negative control levels [20]. It should be noted that extruded FBFs are fortified with 40 mg/100 g ascorbic acid, and CSB13 and CSB+ are fortified with 40 mg/100 g and 90 mg/100 g ascorbic acid, respectively (Table 3). Some studies have achieved significant ferritin response by increasing the food sample quantity. One study increased the sample quantity from 0.5 g to 1 g and then to 3 g due to low ferritin response with lower amounts (< 3 g) of food [24]. However, their unleavened bread flour samples contained very low iron Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 concentrations (0.67-4.67 mg/100 g) compared to our FBF samples (8.0-31.8 mg iron/100 g). This experiment used 2 g porridge, because greater than 2 g resulted in thick sample that digested poorly. The FeSO positive control (0.2 µg iron/well) ferritin levels were significantly higher than FBFs, Cerelac and negative control except for CSB+ and CSB14. This positive control has been used previously [41] although at a 25 µg iron/well treatment, which is greater than 100 times higher than concentration used in this study. However, comparison of our positive control ferritin response with the previous study was not possible be cause the results were presented as percent relative bioavailability compared to the FeSO Aqueous fraction iron levels used to treat the Caco-2 cells were not correlated to ferritin synthesis as has been found previously [20, 21, 35]. Iron analysis showed differences between whole and decorticated dry FBF iron levels. All of the whole sorghum-cowpea FBFs, and whole sorghum-soy FBFs had higher iron levels compared to their corresponding decorticated FBFs (Table 4). As iron is distributed in different regions of the grain, including the outer layers, it is not surprising that decortication or dehulling results in lower iron levels [42, 43]. CSB13 had approximately 2-fold higher, (31.8 mg/100 g) and CSB+ had approximately 2-fold lower (8.0 mg/100 g), dry FBF iron levels compared to the extruded FBFs’ (~18.0 mg/100 g). USDA commodity requirements [13, 14], require that CSB13 contain 14.7 to 30.0 mg iron/100 g and CSB+ contain 9.0 to 21.0 mg iron/100 g. Thus, iron levels in CSB13 were slightly higher than the upper limit, whereas CSB+ iron levels were slightly below the lower limit set by USDA. All of the extruded FBFs contained 4- to 7-fold significantly higher aqueous fraction iron concentrations compared to CSB13 and CSB+ (Table 4). One of the reasons for the low aqueous fraction iron concentration in CSB13 and CSB+ may be that extruded FBFs were cooked using 20% solids, whereas, CSB13 and CSB+ Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 porridges were cooked using 11.75% and 13.79% solids, per their instructions [13, 14]. There was no difference in aqueous fraction iron levels between CSB13 and CSB+ in spite of 4-fold higher iron levels in dry CSB13 FBF. CSB+ is an improved formulation of CSB13 with enhanced nutrient profile and with ingredients partially cooked through dry roasting [14]. This heat processing partially may explain why CSB+ aqueous fraction iron levels were similar to CSB13; previous studies observed a 16-32% increase in iron availability with roasting and malting [44]. Another reason for the improved iron availability may have been that CSB+ is fortified with both ferrous fumarate and NaFeEDTA. The latter iron fortificant chelates with native iron in the diet and protects it from binding to antinutritional factors [45, 46, 47]. There were no significant differences in Caco-2 cell vitamin A levels (Table 7). Rats fed dry extruded sorghum-cowpea, extruded sorghum- soy and extruded corn-soy (CSB14) FBFs or CSB+ showed no significant difference in serum retinol levels consistent with our Caco -2 results reported here [40]. There is limited data on in-vitro vitamin A bioavailability. While there are many studies on the carotenoid bioavailability [25, 33], to the best of our knowledge only one study has examined vitamin A bioavailability using the Caco -2 cell model [26]. Vitamin A content in the current study ranged from 0.3 mg/100 g in Cerelac to 1.67 mg/100 g in CSB+ (Table 6). Vitamin A content in Cerelac matched its labeled content, however CSB+ and CSB13 had 61% and 41%, respectively, higher vitamin A levels than the required levels (Table 3). Aqueous fraction vitamin A concentrations were not significantly different (Table 6). Interestingly, non-extruded CSB13 and CSB+ that contained highest dry FBF vitamin A levels had the lowest aqueous fraction concentrations compared to extruded FBFs and Cerelac. It is possible, that the longer cooking time for preparing these porridges may have affected the vitamin A concentrations [26]. Another reason for CSB+’s lower vitamin A aqueous fractions levels maybe that it contains lower fat levels than the newly formulated FBFs. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Limitations The major limitation of this study is that we did not find significant differences between the negative control and our treatments. Because this is the first study looking at both bioavailable iron and vitamin A levels, we used a method developed for assessing carotenoid bioavailability [48, 25, 28, 33] and modified it to estimate both iron and vitamin A simultaneously. Since this methodology is new, it has not been validated against human bioavailability data. However, it is notable that the lack of differences in iron and vitamin A outcomes between extruded FBFs is consistent with a rat study that fed these blends [40]. Use of inserts with dialysis membranes in a two-compartment arrangement as has been done previously [18, 19, 20, 21, 23, 49] may have resulted in different outcomes. However, it should be noted that there are previous studies using the same two-compartment methodology have also not found significant changes compared to negative controls [38, 39]. Another possibility might have been spiking the food samples with external iron. This might help improve the ferritin response. Assessing cell differentiation by measuring the brush border enzyme activity [50] rather than the 14-days post seeding like was used in this study may have also improved outcomes. Overall, lack of method standardization and responsiveness to treatment can be viewed as limitations of the in vitro digestion/Caco-2 model. Further studies are recommended using animals and humans to determine the iron and vitamin A bioavailability of new extruded FBFs and traditional non-extruded FBFs. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Conclusions To the best of our knowledge, this is the first study to measure both bioavailable iron and vitamin A using in-vitro digestion/Caco-2 cell model. While the lack of difference between the negative control and treatments should be noted, our results suggest that consumption of newly developed extruded sorghum-cowpea, sorghum-soy, and corn-soy FBFs will result in iron and vitamin A status comparable to traditional non-extruded CSB13 and CSB+ FBFs. Acknowledgments This study was supported by the United States Department of Agriculture Micronutrient Fortified Food Aid Products Pilot Progr am (MFFAPP), contract number #FFE-621-2012/033-00. This is contribution no. 17-019-J of the Kansas Agricultural Experiment Station, Manhattan, KS. The authors would like to thank Grant Geiger for his technical assistance. Author’s contributions to manuscript: B.L. and S.A. conceived and designed the experiments; K.P. performed the experiments; K.P. N.F. analyzed the data; K.P. and B.L. contributed reagents/materials/analysis tools; K.P. wrote the paper. All authors read and approved the final manuscript. References 1. Fleige LE, Moore WR, Garlick PJ, Murphy SP, Turner EH, Dunn ML, van Lengerich B, Orthoefer FT, Schaefer SE. Recommendations for optimization of fortified and blended food aid products from the United States. Nutr Rev. 2010;68: 290-315. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 2. Webb P, Rogers B, Rosenberg I, Schlossman N, Wanke C, Bagriansky J, Sadler K, Johnson Q, Tilahun J, Masterson AR, Narayan A. 2011. Delivering improved nutrition: recommendations for changes to US food aid products and programs. Boston, MA: Tufts University. 3. UN, Food and Agricultural Organization. Sorghum. Crops. FAOSTAT Domains. Production [Internet]. 2013 [cited 2016 August 16]. Available from: http://faostat3.fao.org/browse/Q/*/E. 4. Henley EC, Rooney L, Dahlberg J, Bean S, Weller C, Turner N, Awika J, Haub M, Smail V. Sorghum: an ancient, healthy and nutri tious old world cereal. The United Sorghum Checkoff Program [Internet]. 2010 [cited 2017 December 20]. Available from: http://sorghum.ucanr.edu/data/files/Sorghum%20Ancient%20Grain%20Final%209-16-10.pdf. 5. Akibode S, Maredia M. Global and regional trends in production, trade and consumption of food legume crops. Report Submitted to SPIA. Department of Agricultural, Food and Resource Economics, Michigan State University [Internet]. 2011 [cited 2017 December 20]. Available from: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.374.3946&rep=rep1&type=pdf. 6. Kayodé AP, Linnemann AR, Hounhouigan JD, Nout MJ, van Boekel MA. Genetic and environmental impact on iron, zinc, and phytate in food sorghum grown in Benin. J Agric Food Chem. 2006;54: 256-262. 7. Punia KP, Darshan. Proximate composition, phytic acid, polyphenols and digestibility (in-vitro) of four brown cowpea varieties. Int J Food Sci Nutr. 2000;51: 189-193. 8. Radhakrishnan MR, Sivaprasad J. Tannin content of sorghum varieties and their role in iron bioavailability. J Agric Food Chem. 1980;28: 55-57. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 9. Plahar W, Annan N, Nti C. Cultivar and processing effects on the pasting characteristics, tannin content and protein quality and digestibility of cowpea (Vigna unguiculata). Plant Foods Hum Nutr. 1997;51: 343-356. 10. Ilo S, Schoenlechner R, Berghofe E. Role of lipids in the extrusion cooking processes. Grasas Y Aceites. 2000;51: 97-110. 11. Alonso R, Rubio L, Muzquiz M, Marzo F. The effect of extrusion cooking on mineral bioavailability in pea and kidney bean seed meals. Anim Feed Sci Technol. 2001;94: 1-13. 12. Tran QD, Hendriks WH, van der Poel, Antonius FB. Effects of extrusion processing on nutrients in dry pet food. J Sci Food Agric. 2008;88: 1487-1493. 13. USDA Commodity Requirements. Corn-Soy Blend for use in export programs [Internet]. 2014 [cited 2017 Dec 30]. Available from: https://www.fsa.usda.gov/Internet/FSA_File/csb13_110507.pdf. 14. USDA Commodity Requirements. Corn-Soy Blend Plus for Use in International Food Aid Programs [Internet]. 2014 [cited 2017 Dec 30]. Available from: https://www.fsa.usda.gov/Internet/FSA_File/csbp2.pdf. 15. Delimont NM, Chanadang S, Joseph MV, Rockler BE, Guo Q, Regier GK, Mulford GR, Kayanda R, Range M, Mziray Z et al. The MFFAPP Tanzania efficacy study protocol: newly formulated, extruded fortified-blended foods for food aid. Curr Dev Nutr. 2017;1: 1-9. 16. Ahmed T, Hossain M, Sanin KI. Global burden of maternal and child undernutrition and micronutrient deficiencies. Ann Nutr Metab. 2012;61 Suppl 1: 8- 17. Sandberg A. The use of Caco-2 cells to estimate Fe absorption in humans--A critical appraisal. Int J Vitam Nutr Res. 2010;80: 307-313. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 18. Pullakhandam R, Nair KM, Pamini H, Punjal R. Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model. J Food Sci. 2011;76: H38-H42. 19. Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD. Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in-vitro digestion/Caco-2 cell culture model. J Nutr. 1998;128: 1555-1561. 20. Glahn R, Cheng Z, Welch R, Gregorio G. Comparison of iron bioavailability from 15 rice genotypes: studies using an in-vitro digestion/caco-2 cell culture model. J Agric Food Chem. 2002;50: 3586-3591. 21. Viadel B. Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas. Food Chem. 2007;102: 925- 22. Lung'aho MG, Glahn R. In-vitro estimates of iron bioavailability in some Kenyan complementary foods. Food Nutr Bull. 2009;30: 145-152. 23. DellaValle D, Vandenberg A, Glahn R. Seed coat removal improves iron bioavailability in cooked lentils: studies using an in-vitro digestion/Caco-2 cell culture model. J Agric Food Chem. 2013;61: 8084-8089. 24. Eagling T, Wawer A, Shewry P, Zhao F, Fairweather Tait S. Iron bioavailability in two commercial cultivars of wheat: comparison between wholegrain and white flour and the effects of nicotianamine and 2'-deoxymugineic acid on iron uptake into Caco-2 cells. J Agric Food Chem. 2014;62: 10320-10325. 25. Thakkar SK, Maziya-Dixon B, Dixon AG, Failla ML. Beta-carotene micellarization during in-vitro digestion and uptake by Caco-2 cells is directly proportional to beta-carotene content in different genotypes of cassava. J Nutr. 2007;137: 2229-2233. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 26. O’Callaghan Y, O’Brien N. Bioaccessibility, cellular uptake and transepithelial transport of α‐tocopherol and retinol from a range of supplemented foodstuffs assessed using the caco‐2 cell model. Int J Food Sci Tech. 2010;45: 1436-1442. 27. General Food Distribution, Module 11. Part2: Technical Notes. Food basket commodities [Internet]. 2011 [cited 2016 August 16]. Available online: http://www.unscn.org/layout/modules/htp/pdf/M11P2.pdf. 28. Garrett DA, Failla ML, Sarama RJ. Development of an in-vitro digestion method to assess carotenoid bioavailability from meals. J Agric Food Chem. 1999;47: 4301-4309. 29. Jin F, Frohman C, Thannhauser T, Welch R, Glahn R. Effects of ascorbic acid, phytic acid and tannic acid on iron bioavailability from reconstituted ferritin measured by an in-vitro digestion-Caco-2 cell model. Br J Nutr. 2009;101: 972-981. 30. Etcheverry P, Miller D, Glahn R. A low-molecular-weight factor in human milk whey promotes iron uptake by Caco-2 cells. J Nutr. 2004;134: 93-98. 31. Liu C, Glahn RP, Liu RH. Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in-vitro digestion. J Agric Food Chem. 2004;52: 4330-4337. 32. Gallardo-Guerrero L, Gandul-Rojas B, Mínguez-Mosquera MI. Digestive stability, micellarization, and uptake by Caco-2 human intestinal cell of chlorophyll derivatives from different preparations of pea (Pisum sativum L.). J Agric Food Chem. 2008;56: 8379-8386. 33. Failla ML, Chitchumronchokchai C, Ferruzzi MG, Goltz SR, Campbell WW. Unsaturated fatty acids promote bioaccessibility and basolateral secretion of carotenoids and α-tocopherol by Caco-2 cells. 2014;5: 1101-1112. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 34. Latunde-Dada GO, Li X, Parodi A, Edwards C, Ellis P, Sharp PA. Micromilling enhances iron bioaccessibility from wholegrain wheat. J Agric Food Chem. 2014;62: 11222-11227. 35. Cilla A, Perales S, Lagarda M, Barbera R, Farre R. Iron bioavailability in fortified fruit beverages using ferritin synthesis by Caco-2 cells. J Agric Food Chem. 2008;56: 8699-8703. 36. Lindshield BL, King JL, Wyss A, Goralczyk R, Lu CH, Ford NA, Erdman JW, Jr. Lycopene biodistribution is altered in 15,15'-carotenoid monooxygenase knockout mice. J Nutr. 2008;138: 2367-2371. 37. Deming DM, Teixeira SR, Erdman JW, Jr. All-trans beta-carotene appears to be more bioavailable than 9-cis or 13-cis beta-carotene in gerbils given single oral doses of each isomer. J Nutr. 2002;132: 2700-2708. 38. Tan SY, Yeung CK, Tako E, Glahn RP, Welch RM, Lei X, Miller DD. Iron bioavailability to piglets from red and white common beans (Phaseolus vulgaris). J Agric Food Chem. 2008;56: 5008-5014. 39. Tako E, Beebe SE, Reed S, Hart JJ, Glahn RP. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr J. 2014;13:28. 40. Delimont NM, Fiorentino NM, Opoku-Acheampong AB, Joseph MV, Guo Q, Alavi S, Lindshield BL. Newly formulated, protein qualityenhanced, extruded sorghum-, cowpea-, corn-, soya-, sugar- and oilcontaining fortified-blendedfoodslead to adequatevitamin A and iron outcomesandimprovedgrowthcompared withnon-extrudedCSB+in rats. J Nutr Sci 2017;6:e18. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 41. Proulx AK, Reddy MB. Iron bioavailability of hemoglobin from soy root nodules using a Caco-2 cell culture model. J Agric Food Chem. 2006;54: 1518- 42. Ghavidel RA and Prakash J. The impact of germination and dehulling on nutrients, antinutrients, in-vitro iron and calcium bioavailability and in-vitro starch and protein digestibility of some legume seeds. LWT-Food Sci and Tech. 2007;40: 1292-1299. 43. Lestienne I, Buisson M, Lullien-Pellerin V, Picq C, Trèche S. Losses of nutrients and anti-nutritional factors during abrasive decortication of two pearl millet cultivars (Pennisetum glaucum). Food Chem. 2007;100: 1316-1323. 44. Gahlawat P, and Sehgal S. In-vitro starch and protein digestibility and iron availability in weaning foods as affected by processing methods. Plant Foods Hum Nutr. 1994;45: 165-173. 45. Chang S, Huang Z, Ma Y, Piao J, Yang X, Zeder C, Hurrell RF, Egli I. Mixture of ferric sodium ethylenediaminetetraacetate (NaFeEDTA) and ferrous sulfate: an effective iron fortificant for complementary foods for young Chinese children. Food Nutr Bull. 2012;33: 111-116. 46. Gibson RS, Bailey KB, Gibbs M, Ferguson EL. A review of phytate, iron, zinc, and calcium concentrations in plant-based complementary foods used in low-income countries and implications for bioavailability. Food Nutr Bull. 2010;31: S134-146. 47. Bothwell TH, and MacPhail AP. The potential role of NaFeEDTA as an iron fortificant. Int J Vitam Nutr Res. 2004;74: 421-434. 48. Failla, ML, Huo T, Thakkar, SK. In vitro screening of relative bioaccessibility of carotenoids from foods. Asia Pac. J. Clin. Nutr. 2008; 17(Suppl 1): 200-203. 49. Ariza-Nieto M, Blair MW, Welch RM, Glahn RP. Screening of iron bioavailability patterns in eight bean (Phaseolus vulgaris L.) genotypes using the Caco-2 cell in-vitro model. J Agric Food Chem. 2007;55: 7950-7956. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 50. Jovaní M, Barberá R, Farré R, Martín de Aguilera E. Calcium, iron, and zinc uptake from digests of infant formulas by Caco-2 cells. J Agric Food Chem. 2001;49: 3480-3485. 51. US Food and Drug Administration, Guidance for Industry: A Food Labeling Guide (15. Appendix G: Daily Values for Infants, Children Less Than 4 Years of Age, and Pregnant and Lactating Women. [Internet]. 2013 [updated 2013 January; cited 2018 January 4]. Available from: https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/LabelingNutrition/ucm2006828.htm. Table 1. Extruded fortified blended foods composition Sorghum-Cowpea Sorghum-Soy Corn-Soy Ingredient (%) blend blend blend (SCB, n=7) (SSB, n=3)* (CSB14, n=1) Sorghum flour 24.7 47.6 - Cowpea flour 38.6 - - Corn flour - - 48.1 Soy flour - 15.7 15.2 Sugar 15.0 15.0 15.0 Whey protein concentrate (WPC 80) 9.5 9.5 9.5 Vegetable oil 9.0 9.0 9.0 Vitamin and mineral premix 3.2 3.2 3.1 * For extruded SSB (full-fat soy), WPC80 was increased from 9.5% to 13% and vegetable oil was decreased from 9% to 5.5%. SCB; sorghum-cowpea blend, SSB; sorghum-soy blend, CSB; corn-soy blend. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 2. Non-extruded fortified blended foods and Cerelac compositions Non-Extruded FBFs Cerelac Corn-Soy Corn-Soy Ingredient (%) blend 13 Ingredient blend plus (CSB+) (CSB13) Cornmeal 69.5 - Wheat flour Soy flour, defatted 21.9 - Fat free milk Soybean oil, refined 5.5 - Sugar Minerals 3.0 - Milk fat Vitamin antioxidant premix 0.1 - Corn oil Corn (white or yellow) - 78.5 Palm oil Whole soybeans - 20.0 Calcium carbonate Vitamin/mineral - 0.2 Sodium phosphate Tri-calcium phosphate - 1.2 Bifidus cultures Potassium chloride - 0.2 Vitamin/ mineral For Cerelac, ingredient % was not available, ingredients were listed in the order on the label. FBF; Fortified blended foods, CSB; corn-soy blend. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 3. Fortified blended food and Cerelac macronutrient and micronutrient compositions (100 g) Extruded FBFs Non-extruded FBFs Nutrient Unit SCB/SSB/CSB14 CSB13 CSB+ Cerelac Energy kcal 380 386 380 400 Protein g 17 16 14 13 Fat g 8 9 6 10 †† Vitamin A mg 0.49 0.82 1.04 0.34 Thiamin (B ) mg 0.65 0.61 0.20 0.28 Riboflavin (B ) mg 0.93 0.48 1.4 0.8 Niacin (B ) mg 9.1 6.3 8.0 2.4 Pantothenic Acid (B ) mg 3.7 3.2 1.6 1.3 Vitamin B mg 0.8 0.5 1.0 0.3 Folic Acid (B ) mg 0.09 0.25 0.11 0.05 Vitamin B mg 0.002 0.001 0.002 0.001 Vitamin D mg 0.03 0.005 0.01 0.004 Vitamin E mg 13.2 1.0 8.3 3.0 Vitamin K mg 0.03 0.001 0.03 0.03 Vitamin C mg 40.0 40.0 90.0 16.0 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Calcium mg 279.1 650.0 452.0 427.0 Total Iron (Fe) mg 13.0 10.6 6.5 5.3 Fe (FeSO ) mg 11.0 0.0 4.0 0.0 Fe (NaFeEDTA) mg 2.0 0.0 2.5 0.0 Fe (Ferrous Fumarate) mg 0.0 10.6 4.0 5.3 Iodine mg 0.23 0.0 0.04 0.03 Phosphorus mg 291.0 522.0 290.0 427.0 Potassium mg 163.2 563.0 140.0 NA Zinc mg 5.5 5.9 5.0 2.1 For Cerelac, macronutrient and micronutrient values were calculated based on the % Daily Value (% DV) information provided on the label, and daily values for children under 4-years provided by reference [51]. †† Extruded and non-extruded FBFs contained vitamin A in the form of retinyl palmitate, while Cerelac contained retinyl acetate. SCB; sorghum-cowpea blend, SSB; sorghum-soy blend, CSB; corn-soy blend. NA- not available. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 31 Table 4.Dry fortified blended food, Cerelac and aqueous fraction iron concentrations (mean ± SEM) Aqueous Dry FBF No Cereal Cereal type Legume fraction (mg/100 g) (µg/10 ml) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 19.5 0.93 ± 0.02 2 White Sorghum 1 Decorticated Cowpea 18.6 1.03 ± 0.06 ac 3 White Sorghum 1 Decorticated-C Cowpea 15.1 0.66 ± 0.11 ac 4 White Sorghum 2 Whole Cowpea 19.5 0.68 ± 0.17 ac 5 White Sorghum 2 Decorticated Cowpea 15.9 0.82 ± 0.03 ac 6 Red Sorghum Whole Cowpea 19.1 0.80 ± 0.08 ac 7 Red Sorghum Decorticated Cowpea 15.3 0.62 ± 0.18 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 23.4 0.95 ± 0.06 ac 9 White Sorghum 1 Decorticated Medium-Fat Soy 15.6 0.85 ± 0.05 10 White Sorghum 1 Whole Full-Fat Soy 20.7 0.92 ± 0.03 Corn-Soy blends ac 11 CSB14 Degermed corn-C Medium-Fat Soy 15.6 0.75 ± 0.09 12 CSB13 Cornmeal Defatted Soy Flour 31.8 0.17 ± 0.03 13 CSB+ Whole Corn Whole Soy 8.0 0.14 ± 0.03 Cerelac bc 14 Wheat Wheat Flour NA 11.4 0.42 ± 0.01 Within a column, means without a common superscript are significantly different (p<0.05). Aqueous fractions (n=2) from two different in-vitro digestion experiments (4 replicates). White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. C; Commercial; CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 5. Caco-2 cell ferritin levels following aqueous fraction treatment (n=2, mean ± SEM) Ferritin No Cereal Cereal type Legume (ng/mg) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 6.21 ± 1.68 2 White Sorghum 1 Decorticated Cowpea 4.66 ± 0.05 3 White Sorghum 1 Decorticated-C Cowpea 4.74 ± 0.05 4 White Sorghum 2 Whole Cowpea 6.52 ± 1.83 5 White Sorghum 2 Decorticated Cowpea 5.47 ± 2.05 6 Red Sorghum Whole Cowpea 5.24 ± 1.35 7 Red Sorghum Decorticated Cowpea 4.13 ± 1.01 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 6.51 ± 2.17 9 White Sorghum 1 Decorticated Medium-Fat Soy 5.97 ± 3.06 10 White Sorghum 1 Whole Full-Fat Soy 4.10 ± 1.22 Corn-Soy blends ab 11 CSB14 Degermed corn-C Medium-Fat Soy 6.78 ± 0.64 12 CSB13 Cornmeal Defatted Soy Flour 4.72 ± 0.73 ab 13 CSB+ Whole Corn Whole Soy 7.39 ± 1.76 Cerelac 14 Wheat Wheat Flour NA 4.89± 0.53 Controls Basal salt solution (negative control) 4.75 ± 1.04 ab FeSO (0.1 µg Fe/well) (positive control) 15.87 ± 5.73 FeSO (0.2 µg Fe/well) (positive control) 29.65 ± 3.26 Within a column, means without a common superscript are significantly different (p<0.05). White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full- fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. C; Commercial, CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 6. Dry fortified blended food, Cerelac and aqueous fraction vitamin A concentrations (n=2, mean ± SEM) Aqueous Dry FBF No Cereal Cereal type Legume fraction (mg/100 g) (ng/ml) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 0.75 60.8 ± 10.0 2 White Sorghum 1 Decorticated Cowpea 0.70 69.6 ± 15.9 3 White Sorghum 1 Decorticated-C Cowpea 0.54 50.8 ± 5.0 4 White Sorghum 2 Whole Cowpea 0.71 52.7 ± 7.7 5 White Sorghum 2 Decorticated Cowpea 0.56 80.1 ± 15.4 6 Red Sorghum Whole Cowpea 0.76 77.6 ± 2.8 7 Red Sorghum Decorticated Cowpea 0.54 67.4 ± 27.7 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 0.72 48.0 ± 4.0 9 White Sorghum 1 Decorticated Medium-Fat Soy 0.59 44.8 ± 10.9 10 White Sorghum 1 Whole Full-Fat Soy 0.75 49.1 ± 0.8 Corn-Soy blends 11 CSB14 Degermed corn-C Medium-Fat Soy 0.56 45.9 ± 12.8 12 CSB13 Cornmeal Defatted Soy Flour 1.16 33.0 ± 0.4 13 CSB+ Whole Corn Whole Soy 1.67 41.5 ± 28.2 Cerelac 14 Wheat Wheat Flour NA 0.30 55.6 ± 25.6 White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. No statistically significant differences. C; Commercial, CSB; corn-soy blend, NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 7. Caco-2 cell vitamin A concentrations following aqueous fraction treatment (n=2, mean ± SEM) Vitamin A No Cereal Cereal type Legume (µg/g cells) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 2.59 ± 0.35 2 White Sorghum 1 Decorticated Cowpea 2.52 ± 0.13 3 White Sorghum 1 Decorticated-C Cowpea 2.34 ± 0.15 4 White Sorghum 2 Whole Cowpea 2.23 ± 0.12 5 White Sorghum 2 Decorticated Cowpea 2.29 ± 0.15 6 Red Sorghum Whole Cowpea 1.89 ± 0.52 7 Red Sorghum Decorticated Cowpea 1.99 ± 0.14 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 1.96 ± 0.11 9 White Sorghum 1 Decorticated Medium-Fat Soy 2.13 ± 0.01 10 White Sorghum 1 Whole Full-Fat Soy 2.09 ± 0.69 Corn-Soy blends 11 CSB14 Degermed corn-C Medium-Fat Soy 1.96 ± 0.01 †† 12 CSB13 Cornmeal Defatted Soy Flour 2.03 †† 13 CSB+ Whole Corn Whole Soy 2.43 Cerelac 14 Wheat Wheat Flour NA 2.24 ± 0.10 Control Basal salt solution (negative control) 2.09 ± 0.21 White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). No significant differences. CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. †† For CSB13 and CSB+ (n=1) was included due to sample loss. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 C; Commercial; CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Developments in Nutrition Oxford University Press

Bioavailable Iron and Vitamin A in Newly Formulated, Extruded Corn, Soybean, Sorghum and Cowpea Fortified-Blended Foods in the In-vitro Digestion/Caco-2 Cell Model

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© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society for Nutrition.
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Abstract

Bioavailable Iron and Vitamin A in Newly Formulated, Extruded Corn, Soybean, Sorghum and Cowpea Fortified-Blended Foods in the In-vitro Digestion/Caco-2 Cell Model 1 1 2, 1 Kavitha Penugonda , Nicole Fiorentino , Sajid Alavi and Brian L. Lindshield * Department of Food, Nutrition, Dietetics and Health. Kansas State University, Manhattan, KS 66506, USA Department of Grain Science and Industry, Manhattan, KS 66506, USA * Corresponding author: Brian Lindshield e-mail: blindsh@k-state.edu Tel.: +1-785-532-7848 Fax: +1-785-532-3132. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Abbreviations used: BHT, butylated hydroxytoluene; CSB, corn-soy blend; CSB+, corn-soy blend plus; FAQR, food aid quality review; FBFs, Fortified blended foods; FeSO , ferrous sulfate; KOH, potassium hydroxide; MFFAPP, Micronutrient Fortified Food Aid Products Pilot Program; NaFeEDTA, sodiu m iron EDTA; SCB, sorghum-cowpea blend; SSB, sorghum-soy blend; USAID, United States Agency for International Development; USDA, United States Department of Agriculture; WPC 80, whey protein concentrate with 80% protein content; Sources of financial support: This study was supported by the United States Department of Agriculture Micronutrient Fortified Food Aid Products Pilot Program (MFFAPP), contract number #FFE-621-2012/033-00. This is contribution no. 17-019-J of the Kansas Agricultural Experiment Station, Manhattan, KS. Conflict of Interest: The authors declare no conflicts of interest. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Abstract Background: Fortified blended foods (FBFs), particularly corn-soybean blend (CSB), are food aid products distributed in developing countries. The United States Agency for International Development (USAID) food aid quality review recommended developing extruded FBFs using alternative commodities such as sorghum. Objective: The objective of the study was to determine bioavailable iron and vitamin A levels, from newly developed, extruded corn, soybean, sorghum, and cowpea FBFs compared to the non-extruded traditional food aid FBFs, corn-soy blend 13 and corn-soy blend plus (CSB+). Methods: Eleven extruded FBFs; sorghum-cowpea (n=7), sorghum-soy (n=3), and corn-soy (n=1), along with two non-extruded FBFs; corn-soy blend 13 (CSB13) and corn-soy blend plus (CSB+), and Cerelac, a commercially available fortified infant food, were prepared. Bioavailable iron and vitamin A levels were assessed using the in-vitro digestion/Caco-2 cell model. Dry FBFs, aqueous fractions, and Caco-2 cell pellets vitamin A levels were analyzed by HPLC. Dry FBFs and aqueous fraction iron levels were measured by atomic absorptiometry and bioavailab le iron assessed by measuring Caco-2 ferritin levels via ELISA. Results: Iron and vitamin A content in Cerelac and dry FBFs ranged from 8.0 to 31.8 mg/100g and 0.3 to 1.67 mg/100g, respectively. All of the extruded FBFs contained 4- to 7-fold significantly higher (p<0.05) aqueous fraction iron concentrations compared to CSB13 and CSB+. However, Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 there were no significant differences in Caco-2 cell ferritin and vitamin A levels between extruded FBFs, non-extruded FBFs, or the basal salt solution negative control. Conclusions: Results support that consumption of newly developed extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs would result in iron and vitamin A levels comparable to traditional non-extruded CSB13 and CSB+ FBFs. Keywords: Fortified blended food; corn-soy blend plus; micronutrient bioavailability; iron; vitamin A, whey protein concent rate, FAQR; food aid; Title II foods, in-vitro digestion/Caco-2 cell model. Introduction Fortified blended foods (FBFs) are porridge mixes composed of cereals and legumes that have been milled and fortified with vitamins and minerals. FBFs are major food aid products for young children, women, and other vulnerable groups in developing countries. Historically corn-soy blend (CSB) has been the most widely distributed FBF in a majority of the food aid receiving countries [1]. The United States Agency for International Development (USAID) Food Aid Quality Review (FAQR) recommended developing novel FBFs using cereals that are both culturally and nutritionally acceptable in Africa. It also recommended sorghum as an alternative to corn or wheat and suggests other legumes could be paired with it as alternatives to soy [2]. One logical legume to investigate is cowpea because Africa is the world leading producer of cowpea (95%) in addition to sorghum (41%) [3]. Both sorghum and cowpea are drought-tolerant, sustainable, and not genetically modified (Non-GMO) grains, which is preferred by some food aid recipient nations. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Sorghum and cowpea are rich in iron and are complementary proteins [4, 5], however, sorghum and cowpea also contain the antinutritional factors, phytates, and tannins, which impair iron bioavailability [6-9]. Extrusion is a food processing technique that cooks food using high temperature under high pressure in combination with moisture and mechanical shear [10]. The desirable effects of this cost-effective method are that it decreases viscosity; increases palatability, starch and protein digestibility; and reduces anti-nutritional factor levels thereby potentially improving iron bioavailability [11, 12]. Extruded novel sorghum-cowpea, sorghum-soy, and corn-soy FBFs were developed based on the USAID FAQR recommendations [2] and USDA commodity requirements [13, 14] as part of the Micronutrient Fortified Food Aid Pilot Project [15]. Traditional, non-extruded FBFs, CSB13, and CSB+, were procured to use as comparisons for the newly developed FBFs. The purpose of this study was to assess bioavailable iron and vitamin A levels of newly developed extruded sorghum-cowpea blend (SCB), sorghum-soy blend (SSB), and corn-soy blend (CSB14) FBFs compared to traditional non-extruded FBFs, CSB13 and CSB+, in the in- vitro digestion/Caco-2 cell model. These micronutrients deficiencies were chosen because they are a substantial public health issue for many women and children throughout the world [16]. The in-vitro digestion/Caco-2 model was used because it is a widely used, inexpensive model to study the bioavailability of nutrients from foods and supplements [17-21]. It has been successfully used to screen for iron bioavailability of a variety of complementary foods [22], lentils [23], wheat [24], cassava [25], and supplemented food stuffs [26]. We use the term “bioavailable” to describe the amount of compound in the Caco-2 cells after they were treated with aqueous fractions produced by in-vitro digestion. To the best of our knowledge, this is the first study to use this model to assess both bioavailable iron and vitamin A. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Materials and Methods Chemicals Unless stated otherwise, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). Double deionized water was used for porridge preparation, in-vitro digestion, reagent preparation, and vitamin A extraction. To prevent iron contamination, glassware used in the sample preparation, in-vitro digestion, and iron analysis were acid washed by soaking in 5% nitric acid solution for no less than 12 hours and rinsing with double deionized water before use. Acetonitrile, methanol, chloroform, hexane, and ethanol were HPLC grade. FBF Formulations Extruded sorghum-cowpea (n=7), sorghum-soy (n=3), and corn-soy (n=1) were formulated based on FAQR requirements [2]. Two white (variety 1, Fontanelle 4575; variety 2, 738Y) and one red (217X Burgundy) sorghum varieties, whole or decorticated, were used in producing extruded sorghum-cowpea FBFs and cowpea flour was sourced commercially (Table 1 & Table 2).Extruded sorghum-soy FBFs contained white sorghum variety 1 (Fontanelle 4575), whole or decorticated, with low-fat (1.85%), medium-fat (6.94%), or full-fat (16.93%) soy. Extruded CSB14 was formulated with degermed corn with medium-fat soy. The other FBF components; sugar, oil, whey protein concentrate with 80% protein content (WPC 80), and vitamin-mineral premix were added after extrusion to prevent destruction of micronutrients. Both non-extruded FBFs, CSB13 and CSB+, were purchased from Bunge Milling (St. Louis, MO). The major difference between these two CSBs is that CSB+ is a more recently released CSB with heat-processed corn and soy and improved micronutrient formulation [27]. Cerelac (Nestle, NJ), a commercially available fortified infant food, was purchased from a local store and included as a reference control as has been done previously Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 [22]. It is worth noting that the iron and vitamin A fortificants differed between Cerelac, extruded, and non-extruded FBFs. Extruded FBFs contained ferrous sulfate (FeSO ) and sodium iron EDTA (NaFeEDTA); CSB+ contained ferrous fumarate and NaFeEDTA; CSB13 and Cerelac contained only ferrous fumarate (Table 3). Extruded and non-extruded FBFs contained retinyl palmitate, while Cerelac contained retinyl acetate. All the FBFs were stored at -20°C in zip lock bags covered with aluminum foil. FBF Porridge Preparation Twenty grams dry FBF or Cerelac was slowly added to 80 g of boiling water in a beaker on a hot plate and stirred vigorously for 2 min, removed from hot plate and stirred for another minute. Non-extruded CSB13 and CSB+ were prepared in a similar manner but 11.75 g and 13.79 g dry FBF, respectively, was used and they were cooked for 10 min on a hot plate following the preparation instructions for CSB+ [13, 14]. Porridges were then covered with aluminum foil and kept in water bath at 37°C for 10 min to prevent skin formation. Porridges were then weighed and water lo st during preparation was added back in to bring the final weight to 100 g. Porridges were transferred to 50 ml polypropylene tubes, co vered in aluminum foil, blanketed with nitrogen, sealed and stored at -80°C. Porridges were prepared in duplicate on two different days (4 replicates). Later, these replicates were used for in-vitro digestion/Caco-2 cell experiments. In-vitro Digestion Porridge aliquots were subjected to in-vitro digestion as described previously [28]. Ten ml of basal salt solution (120 mmol/L NaCl, 5 mmol/L KCl, and 6 mmol/L CaCl ) was added to 2.5 grams of thawed FBF in a beaker, homogenized with a laboratory homogenizer for 2 min, and then Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 mixed on a magnetic stir plate for 5 min. Ten ml aliquots of homogenized FBFs were then subjected to three continuous in-vitro digestion phases: 10 min oral, 1-hour gastric, and 2-hour small intestine. Oral Digestion Saliva solution containing 0.9 mg KCl, 0.89 mg NaPO , 0.57 mg NaSO , 0.3 mg NaCl, and 1.69 mg NaHCO /ml deionized water was prepared 4 4 3 and used for all experiments. Ten ml of homogenized FBF solution and 8 ml of freshly prepared artificial saliva [(uric acid (0.015 mg/ml), urea (0.2 mg/ml), mucin (0.025 mg/ml), and α–amylase (10.55 mg/ml) dissolved in saliva solution)] were added to a 50 ml conical tube. Tubes were mixed well, blanketed with nitrogen, sealed with parafilm and incubated placing horizontally in a shaking water bath at 37°C, 85 rp m for 10 min in the dark. Gastric Digestion After oral digestion, digesta pH was decreased to 2.5 ± 0.1 by slowly adding 1M HCl and then 2 ml of freshly made pepsin solution (40 mg/ml in 100mM HCl) was added. The final volume was then adjusted to 40 ml with basal salt solution, blanketed with nitrogen, sealed with parafilm and incubated in a shaking water bath at 37°C, 85 rpm for 1 hour in the dark. Small Intestinal Digestion The gastric phase was terminated by increasing digesta pH to 6.0 ± 0.1 with 1M NaHCO and placing the tubes on ice. Two ml of pancreatin (10 mg/ml) and lipase (5 mg/ml) solution (both in in 100mM NaHCO ) was then added along with 3 ml of bile extract (40 mg/ml 100mM NaHCO ) and the digesta pH was 3 3 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 adjusted to 6.5 ± 0.1 with 1 M NaOH. The final volume was adjusted to 50 ml with basal salt solution, blanketed with nitrogen, sealed with parafilm and incubated in a shaking water bath at 37°C, 85 rpm for 2 hours in the dark. Isolation of Aqueous Fraction from Digesta After small intestine digestion, 10 ml digesta aliquots were transferred to 15 ml polypropylene tubes and centrifuged at 5000 g for 45 min at 5°C. Supernatants were collected by puncturing the side of the tube with an 18 gauge needle and 10 ml syringe without disturbing the pellet. Supernatants were filtered using 0.22 µm syringe filters (SLGP 033 RS, Millipore, MA), and fresh aqueous fractions were used to treat the Caco-2 cells. Aqueous fractions aliquots were blanketed with nitrogen and stored at -80°C for later analysis [28]. Dry Porridge Blends and Aqueous Fractions Iron Determination Dry porridge blend and aqueous fraction iron content were analyzed using an atomic absorption spectrophotometer (AAS). Dry po rridge blends’ iron levels were analyzed (AACC 40-70, 1999) by American Institute of Baking International Analytical Services (Manhattan, KS). Briefly, ten grams of sample was taken in ashing vessels and dried to ash overnight at 500°C in a muffle furnace. Residue was dissolved in 10 ml of concentrated HCl, boiled and evaporated to near dryness on a hot plate. Resulting residue was redissolved in 20 ml of 2N HCl, filtered and diluted to 100 ml with water. Iron concentrations were then measured on an atomic absorption spectrophotometer. Aqueous fractions’ iron concentratio ns, filtered samples, were directly measured on an AAS (Perkin Elmer, AAnalyst 100). Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Caco-2 Cell Cultures Caco-2 cells (ATCC® HTB37™) purchased from American Type Culture Collection (Manassas, VA) were used in the experiment at passage 32 and 33. The cells were maintained at 37°C in an incubator with a 5% CO /95% humidity, and media was changed every other day. Caco-2 cells were initially cultured in growth-enhanced treated T-75 flasks (TP 90076, Midsci, MO) in the presence of Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Grand Island, NY), supplemented with 15% fetal bovine serum (FBS, Atlanta Biologicals, GA), 1% L-glutamine, 1% non-essential amino acids, 1% antibiotic/antimycotic (penicillin/streptomycin) solution, and 0.2% amphotericin B [28]. Confluent cells were subcultured by incubating with 5 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) solution for 5 min, which was then inactivated by adding 10 ml of 15% DMEM. After trypsinization, cell suspensions were collected into 50 ml conical tubes, centrifuged at 800 rpm for 5 min at room temperature, the supernatant media discarded and the cell pellet collected. After re-suspending the cell pellet and counting with a hemocytometer, cells were seeded at 50,000 cells/cm in tissue culture treated 6-well plates (Corning Inc, Corning, NY). After being seeded at day 0, the cells usually became confluent 4-5 days later, at which point they were switched from media containing 15% FBS to 7.5% FBS to slow growth. Cells were used in the iron and vitamin A bioavailability experiments 14 days post-seeding [29, 30]. Aqueous Fraction Caco-2 Treatment On day 13, a day before the experiment, Caco-2 monolayers were provided fresh media. On day 14, media was removed before treating the cells with 0.25 ml fresh aqueous fraction and 1.75 ml of DMEM for iron or 0.5 ml fresh aqueous fraction and 1.5 ml of DMEM for vitamin A, which were then incubated for 12 [31, 32] or 4 hours [33, 26, 25], respectively. Samples were randomly assigned to wells; Cerelac was used as a reference Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 control on each plate. A negative control was prepared with 0.25 ml of basal salt solution containing no iron and 1.75 ml of DMEM. A positive control was prepared with basal salt solution to provide 0.1 µg iron/well or 0.2 µg iron/well. These iron concentrations were selected to match with the estimated iron concentration in the digested FBF aliquots that were added to the Caco-2 cells. In-vitro digestion and Caco-2 cell culture experiments were completed in duplicate on different days using different cells passages. Caco-2 Ferritin and Protein Determination After incubation, treatments were removed and cells were washed with 2 ml of ice cold 2X PBS. Caco -2 monolayers were lysed by adding 350 µl/well of mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific, Rockford, IL) [34] and incubated in 6-well plates for 10 minutes on a plate shaker at 120 rpm. Caco-2 monolayers were scraped with a cell scraper (Fisher Scientific, Pittsburgh, PA), collected into microcentrifuge tubes, sonicated for 3 minutes and centrifuged at 14,000g for 10 minutes. Cell lysate supernatants were transferred to microcentrifuge tubes and stored at -20°C for ferritin and protein determination, which was completed within 24 hours [34, 35, 21]. Ten µl cell lysate solutions were used for determining ferritin concentrations (ng/ml) using enzyme-linked immunosorbent assay (ELISA, Spectro Ferritin kit, S- 22, Ramco Laboratories Inc., Stafford, TX) as done previously [23, 35]. Twenty five µl cell lysate solutions were used for measuring protein concentrations using Pierce bicinchoninic acid protein assay kits (Rockford, IL). Ferritin content (ng/mg cell protein) was c alculated as a ratio of cell ferritin (ng/ml)/cell protein (mg/ml) [35]. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Harvesting Caco-2 Monolayers for Vitamin A Assessment After incubation, treatments were removed and cells were washed with 2 ml of ice cold 2X PBS followed by 2 ml of ice cold 2 g /L albumin in PBS. After discarding the washing solutions, Caco-2 monolayers were removed using a cell scraper (Fisher Scientific, Pittsburgh, PA), and collected into amber colored microcentrifuge tubes using 1 ml ice cold PBS. This process was repeated twice more using 0.5 ml ice cold PBS, for a total of 2 ml PBS collected into the same microcentrifuge tubes and centrifuged at 2000 rpm for 45 minutes at 5°C. Supernatant PBS was discarded by carefully inverting the microcentrifuge tubes for 20 seconds. Tubes with cell pellets were then blanketed with nitrogen, and stored at -80°C for vitamin A analysis [33]. Extraction of Vitamin A in Dry Porridge, Aqueous Fraction and Caco-2 Cells Vitamin A was extracted from dry porridge, aqueous fractions and Caco-2 cell pellets as described previously [36] with modifications. For dry porridge, approximately 1 g of dry porridge in a 50 ml screw cap glass tube was homogenized in 4 ml of deionized water before adding 10 ml of ethanol with 0.1% butylated hydroxytoluene (BHT) and 4 mL of super saturated potassium hydroxide (KOH) solution. Samples were vortexed and incubated in a water bath at 70°C for 30 minutes, vortexing every 10 minutes. The tubes were placed on ice and 6 ml of deionized distilled water was added. The samples were initially extracted with 10 ml of hexane and then twice with 5 ml of hexane. Tubes were vortexed each time hexane was added and left on ice to allow layer separation. The top hexane layer was collected into a clean glass tube with Pasteur pipette, completely dried in vacufuge (Model No, 5301, Eppendorf North America, Hauppauge, NY, USA), and reconstituted in 400 µl of mobile phase. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 For aqueous fractions, 8 ml of the thawed aqueous fraction was extracted with 10 ml of ethanol with 0.1% BHT and 4 mL of supe r saturated KOH. The rest of the extraction procedure was the same as described for dry porridge. Finally, reconstituted (in 400 µl mobile phase) aqueous fractions were syringe filtered using Whatman PVDF filters with 0.45µm pore size (Cat No. 6779-1304, GE Healthcare Biosciences, Pittsburgh, PA, USA) before placing in HPLC vials. Cell pellets were weighed then 2 ml of ethanol with 0.1% BHT and 1 mL of super saturated KOH were added and incubated at 70°C for 30 minutes, vortexing every 10 minutes [26]. The tubes were kept on ice and 1 ml of deionized distilled water was added. Samples were extracted with 5 ml of hexane three times and the hexane layers were dried in the manner similar to dry porridges. Dried hexane layers were reconstituted in 400 µl of mobile phase. HPLC Analysis All samples were analyzed the same day they were extracted. A Shimadzu HPLC system (Kyoto, Japan) consisting of a DGU-20A3 built in degasser, a LC-20AB solvent delivery pump, a SIL-20ACHT auto-sampler, a CTO-20AC column holding oven, a CBM-20A communicator module, and a SPD-M20A Photodiode Array Detector with an Agilent Eclipse XDB 5mm C18 (250mm x 4.6mm, Santa Clara, CA, USA) analytical column 25°C was used for analysis. A mobile phase of methanol/acetonitrile/chloroform (47:47:6, v/v/v) at a flow rate of 1.0 mL/min was used, detection was at 325 nm [37] and data was analyzed using LC solution software (Kyoto, Japan). An external retinyl acetate (catalog # 1716002, USP, Rockville, MD, USA) standard curve was used for quantification, with its concentration determined using a spectrophotometer ( Jenway 6305, Bibby Scientific US, NJ, USA). Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Statistical Analysis Data were analyzed using one-way ANOVA with Tukey’s test on SAS 9.3 (SAS Institute Inc., Cary, NC, USA), with p < 0.05 considered significant. Natural logs were used to transform data that did not meet the model assumptions. Results Iron Concentration in Dry FBF and Aqueous Fraction Dry FBFs’ iron concentration ranged from 8.0 to 31.8 mg/100 g (Table 3). Extruded dry FBF iron concentrations (15.1 to 23.4 mg/100 g) were higher than CSB+ (8.0 mg/100 g), but lower than CSB13 (31.8 mg/100 g). Cerelac’s iron concentration (11.0 mg/100 g) was lower than all the FBFs except CSB+. Among the sorghum-containing FBFs, iron levels were higher in whole sorghum compared to the decorticated FBFs. Aqueous fractions iron levels ranged from 0.14 to 1.03 µg/ml (Table 3). All extruded FBFs had significantly higher aqueous fractions iron levels compared to CSB13 and CSB+. There were no significant differences between extruded FBFs or between CSB13, CSB+, and Cerelac aqueous fraction levels. Cerelac aqueous fraction iron levels (0.42 µg/ml) were significantly lower compared to whole white sorghum1 -cowpea, decorticated white sorghum1-cowpea, whole sorghum-soy (low-fat), and whole sorghum-soy (full-fat) extruded FBFs. Ferritin Concentration in Caco-2 Cells Treated With Aqueous Fractions There were no significant differences between FBF aqueous fraction and basal salt solution (negative control) treatment ferritin levels (Table 5). Among the extruded FBFs, CSB14 had the highest ferritin levels (6.78 ng/mg) and whole sorghum-soy (full-fat) had the lowest ferritin levels (4.10 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 ng/mg). The FeSO positive control (0.2 µg iron/well) treatment resulted in significantly higher ferritin levels than Cerelac, negative co ntrol and all FBFs except for corn-soy (medium-fat) and CSB+. There was a dose-dependent increase in ferritin levels in response to FeSO treatment. Vitamin A Concentration in Dry FBF and Aqueous Fraction Dry FBFs’ vitamin A concentrations ranged from 0.54-1.67 mg/100 g (Table 6). CSB+ (1.67 mg/100 g) and Cerelac (0.3 mg vitamin A/100 g) had the highest and lowest vitamin A concentrations, respectively. Whole sorghum FBFs had slightly higher vitamin A levels co mpared to their corresponding decorticated sorghum FBFs. FBFs’ and Cerelac’s aqueous fraction vitamin A concentrations were similar and not significantly different. However, in general, sorghum-cowpea FBFs contained higher levels (50.8-80.1 ng/ml) than sorghum-soy and corn-soy FBFs (33.0-49.1 ng/ml). Vitamin A Concentration in Caco-2 Cells Treated With Aqueous Fraction Caco-2 cell pellet vitamin A concentrations were not significantly different, following aqueous fraction treatment, between extruded, non-extruded FBFs and Cerelac (Table 7). The interesting trend was that red sorghum levels were lower than white sorghum. Sorghum-soy FBFs with full-fat and medium-fat soy had nonsignificantly higher vitamin A levels than low-fat sorghum-soy FBF. The vitamin A levels in all the FBFs and Cerelac are higher than the negative control levels of 2.09 µg/g, with the exception of red sorghum FBFs, white whole sorghum-soy (low-fat soy), CSB13, and CSB14. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Discussion Seven of the extruded FBFs and one non-extruded FBF had ferritin response higher than negative control levels, but none were significantly higher (Table 5). It should be noted that previous studies conducted with similar food matrix have also not observed increases above negative control ferritin response. In a study comparing standard and iron biofortified black beans using the in vitro digestion/Caco-2 model, ferritin response for black bean varieties was significantly lower than the negative control [39]. In another study comparing red and white beans using the in vitro digestion/Caco-2 model, all sample ferritin responses were at, or significantly below, the negative control [38]. It should also be noted that the same manuscript found that white bean consumption resulted significant improvements in iron status compared to red bean consumptio n in pigs [38]. Rats fed dry extruded sorghum-cowpea, extruded sorghum-soy and extruded corn-soy (CSB14) FBFs or CSB+ showed no significant difference in hemoglobin and liver iron levels consistent with our Caco-2 results reported here [40]. The amount of iron added to Caco-2 cells in each well ranged from 0.11-0.25 µg/well, which was similar to the range (0.05- 0.39 µg/well) found in different rice varieties that also did not significantly increase ferritin levels [20]. However, adding ascorbic acid, iron bioavailability of the rice sample significantly increased beyond the negative control levels [20]. It should be noted that extruded FBFs are fortified with 40 mg/100 g ascorbic acid, and CSB13 and CSB+ are fortified with 40 mg/100 g and 90 mg/100 g ascorbic acid, respectively (Table 3). Some studies have achieved significant ferritin response by increasing the food sample quantity. One study increased the sample quantity from 0.5 g to 1 g and then to 3 g due to low ferritin response with lower amounts (< 3 g) of food [24]. However, their unleavened bread flour samples contained very low iron Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 concentrations (0.67-4.67 mg/100 g) compared to our FBF samples (8.0-31.8 mg iron/100 g). This experiment used 2 g porridge, because greater than 2 g resulted in thick sample that digested poorly. The FeSO positive control (0.2 µg iron/well) ferritin levels were significantly higher than FBFs, Cerelac and negative control except for CSB+ and CSB14. This positive control has been used previously [41] although at a 25 µg iron/well treatment, which is greater than 100 times higher than concentration used in this study. However, comparison of our positive control ferritin response with the previous study was not possible be cause the results were presented as percent relative bioavailability compared to the FeSO Aqueous fraction iron levels used to treat the Caco-2 cells were not correlated to ferritin synthesis as has been found previously [20, 21, 35]. Iron analysis showed differences between whole and decorticated dry FBF iron levels. All of the whole sorghum-cowpea FBFs, and whole sorghum-soy FBFs had higher iron levels compared to their corresponding decorticated FBFs (Table 4). As iron is distributed in different regions of the grain, including the outer layers, it is not surprising that decortication or dehulling results in lower iron levels [42, 43]. CSB13 had approximately 2-fold higher, (31.8 mg/100 g) and CSB+ had approximately 2-fold lower (8.0 mg/100 g), dry FBF iron levels compared to the extruded FBFs’ (~18.0 mg/100 g). USDA commodity requirements [13, 14], require that CSB13 contain 14.7 to 30.0 mg iron/100 g and CSB+ contain 9.0 to 21.0 mg iron/100 g. Thus, iron levels in CSB13 were slightly higher than the upper limit, whereas CSB+ iron levels were slightly below the lower limit set by USDA. All of the extruded FBFs contained 4- to 7-fold significantly higher aqueous fraction iron concentrations compared to CSB13 and CSB+ (Table 4). One of the reasons for the low aqueous fraction iron concentration in CSB13 and CSB+ may be that extruded FBFs were cooked using 20% solids, whereas, CSB13 and CSB+ Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 porridges were cooked using 11.75% and 13.79% solids, per their instructions [13, 14]. There was no difference in aqueous fraction iron levels between CSB13 and CSB+ in spite of 4-fold higher iron levels in dry CSB13 FBF. CSB+ is an improved formulation of CSB13 with enhanced nutrient profile and with ingredients partially cooked through dry roasting [14]. This heat processing partially may explain why CSB+ aqueous fraction iron levels were similar to CSB13; previous studies observed a 16-32% increase in iron availability with roasting and malting [44]. Another reason for the improved iron availability may have been that CSB+ is fortified with both ferrous fumarate and NaFeEDTA. The latter iron fortificant chelates with native iron in the diet and protects it from binding to antinutritional factors [45, 46, 47]. There were no significant differences in Caco-2 cell vitamin A levels (Table 7). Rats fed dry extruded sorghum-cowpea, extruded sorghum- soy and extruded corn-soy (CSB14) FBFs or CSB+ showed no significant difference in serum retinol levels consistent with our Caco -2 results reported here [40]. There is limited data on in-vitro vitamin A bioavailability. While there are many studies on the carotenoid bioavailability [25, 33], to the best of our knowledge only one study has examined vitamin A bioavailability using the Caco -2 cell model [26]. Vitamin A content in the current study ranged from 0.3 mg/100 g in Cerelac to 1.67 mg/100 g in CSB+ (Table 6). Vitamin A content in Cerelac matched its labeled content, however CSB+ and CSB13 had 61% and 41%, respectively, higher vitamin A levels than the required levels (Table 3). Aqueous fraction vitamin A concentrations were not significantly different (Table 6). Interestingly, non-extruded CSB13 and CSB+ that contained highest dry FBF vitamin A levels had the lowest aqueous fraction concentrations compared to extruded FBFs and Cerelac. It is possible, that the longer cooking time for preparing these porridges may have affected the vitamin A concentrations [26]. Another reason for CSB+’s lower vitamin A aqueous fractions levels maybe that it contains lower fat levels than the newly formulated FBFs. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Limitations The major limitation of this study is that we did not find significant differences between the negative control and our treatments. Because this is the first study looking at both bioavailable iron and vitamin A levels, we used a method developed for assessing carotenoid bioavailability [48, 25, 28, 33] and modified it to estimate both iron and vitamin A simultaneously. Since this methodology is new, it has not been validated against human bioavailability data. However, it is notable that the lack of differences in iron and vitamin A outcomes between extruded FBFs is consistent with a rat study that fed these blends [40]. Use of inserts with dialysis membranes in a two-compartment arrangement as has been done previously [18, 19, 20, 21, 23, 49] may have resulted in different outcomes. However, it should be noted that there are previous studies using the same two-compartment methodology have also not found significant changes compared to negative controls [38, 39]. Another possibility might have been spiking the food samples with external iron. This might help improve the ferritin response. Assessing cell differentiation by measuring the brush border enzyme activity [50] rather than the 14-days post seeding like was used in this study may have also improved outcomes. Overall, lack of method standardization and responsiveness to treatment can be viewed as limitations of the in vitro digestion/Caco-2 model. Further studies are recommended using animals and humans to determine the iron and vitamin A bioavailability of new extruded FBFs and traditional non-extruded FBFs. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Conclusions To the best of our knowledge, this is the first study to measure both bioavailable iron and vitamin A using in-vitro digestion/Caco-2 cell model. While the lack of difference between the negative control and treatments should be noted, our results suggest that consumption of newly developed extruded sorghum-cowpea, sorghum-soy, and corn-soy FBFs will result in iron and vitamin A status comparable to traditional non-extruded CSB13 and CSB+ FBFs. Acknowledgments This study was supported by the United States Department of Agriculture Micronutrient Fortified Food Aid Products Pilot Progr am (MFFAPP), contract number #FFE-621-2012/033-00. This is contribution no. 17-019-J of the Kansas Agricultural Experiment Station, Manhattan, KS. The authors would like to thank Grant Geiger for his technical assistance. Author’s contributions to manuscript: B.L. and S.A. conceived and designed the experiments; K.P. performed the experiments; K.P. N.F. analyzed the data; K.P. and B.L. contributed reagents/materials/analysis tools; K.P. wrote the paper. All authors read and approved the final manuscript. References 1. Fleige LE, Moore WR, Garlick PJ, Murphy SP, Turner EH, Dunn ML, van Lengerich B, Orthoefer FT, Schaefer SE. Recommendations for optimization of fortified and blended food aid products from the United States. Nutr Rev. 2010;68: 290-315. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 2. Webb P, Rogers B, Rosenberg I, Schlossman N, Wanke C, Bagriansky J, Sadler K, Johnson Q, Tilahun J, Masterson AR, Narayan A. 2011. Delivering improved nutrition: recommendations for changes to US food aid products and programs. Boston, MA: Tufts University. 3. UN, Food and Agricultural Organization. Sorghum. Crops. FAOSTAT Domains. Production [Internet]. 2013 [cited 2016 August 16]. Available from: http://faostat3.fao.org/browse/Q/*/E. 4. Henley EC, Rooney L, Dahlberg J, Bean S, Weller C, Turner N, Awika J, Haub M, Smail V. Sorghum: an ancient, healthy and nutri tious old world cereal. The United Sorghum Checkoff Program [Internet]. 2010 [cited 2017 December 20]. Available from: http://sorghum.ucanr.edu/data/files/Sorghum%20Ancient%20Grain%20Final%209-16-10.pdf. 5. Akibode S, Maredia M. Global and regional trends in production, trade and consumption of food legume crops. Report Submitted to SPIA. Department of Agricultural, Food and Resource Economics, Michigan State University [Internet]. 2011 [cited 2017 December 20]. Available from: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.374.3946&rep=rep1&type=pdf. 6. Kayodé AP, Linnemann AR, Hounhouigan JD, Nout MJ, van Boekel MA. Genetic and environmental impact on iron, zinc, and phytate in food sorghum grown in Benin. J Agric Food Chem. 2006;54: 256-262. 7. Punia KP, Darshan. Proximate composition, phytic acid, polyphenols and digestibility (in-vitro) of four brown cowpea varieties. Int J Food Sci Nutr. 2000;51: 189-193. 8. Radhakrishnan MR, Sivaprasad J. Tannin content of sorghum varieties and their role in iron bioavailability. J Agric Food Chem. 1980;28: 55-57. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 9. Plahar W, Annan N, Nti C. Cultivar and processing effects on the pasting characteristics, tannin content and protein quality and digestibility of cowpea (Vigna unguiculata). Plant Foods Hum Nutr. 1997;51: 343-356. 10. Ilo S, Schoenlechner R, Berghofe E. Role of lipids in the extrusion cooking processes. Grasas Y Aceites. 2000;51: 97-110. 11. Alonso R, Rubio L, Muzquiz M, Marzo F. The effect of extrusion cooking on mineral bioavailability in pea and kidney bean seed meals. Anim Feed Sci Technol. 2001;94: 1-13. 12. Tran QD, Hendriks WH, van der Poel, Antonius FB. Effects of extrusion processing on nutrients in dry pet food. J Sci Food Agric. 2008;88: 1487-1493. 13. USDA Commodity Requirements. Corn-Soy Blend for use in export programs [Internet]. 2014 [cited 2017 Dec 30]. Available from: https://www.fsa.usda.gov/Internet/FSA_File/csb13_110507.pdf. 14. USDA Commodity Requirements. Corn-Soy Blend Plus for Use in International Food Aid Programs [Internet]. 2014 [cited 2017 Dec 30]. Available from: https://www.fsa.usda.gov/Internet/FSA_File/csbp2.pdf. 15. Delimont NM, Chanadang S, Joseph MV, Rockler BE, Guo Q, Regier GK, Mulford GR, Kayanda R, Range M, Mziray Z et al. The MFFAPP Tanzania efficacy study protocol: newly formulated, extruded fortified-blended foods for food aid. Curr Dev Nutr. 2017;1: 1-9. 16. Ahmed T, Hossain M, Sanin KI. Global burden of maternal and child undernutrition and micronutrient deficiencies. Ann Nutr Metab. 2012;61 Suppl 1: 8- 17. Sandberg A. The use of Caco-2 cells to estimate Fe absorption in humans--A critical appraisal. Int J Vitam Nutr Res. 2010;80: 307-313. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 18. Pullakhandam R, Nair KM, Pamini H, Punjal R. Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model. J Food Sci. 2011;76: H38-H42. 19. Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD. Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in-vitro digestion/Caco-2 cell culture model. J Nutr. 1998;128: 1555-1561. 20. Glahn R, Cheng Z, Welch R, Gregorio G. Comparison of iron bioavailability from 15 rice genotypes: studies using an in-vitro digestion/caco-2 cell culture model. J Agric Food Chem. 2002;50: 3586-3591. 21. Viadel B. Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas. Food Chem. 2007;102: 925- 22. Lung'aho MG, Glahn R. In-vitro estimates of iron bioavailability in some Kenyan complementary foods. Food Nutr Bull. 2009;30: 145-152. 23. DellaValle D, Vandenberg A, Glahn R. Seed coat removal improves iron bioavailability in cooked lentils: studies using an in-vitro digestion/Caco-2 cell culture model. J Agric Food Chem. 2013;61: 8084-8089. 24. Eagling T, Wawer A, Shewry P, Zhao F, Fairweather Tait S. Iron bioavailability in two commercial cultivars of wheat: comparison between wholegrain and white flour and the effects of nicotianamine and 2'-deoxymugineic acid on iron uptake into Caco-2 cells. J Agric Food Chem. 2014;62: 10320-10325. 25. Thakkar SK, Maziya-Dixon B, Dixon AG, Failla ML. Beta-carotene micellarization during in-vitro digestion and uptake by Caco-2 cells is directly proportional to beta-carotene content in different genotypes of cassava. J Nutr. 2007;137: 2229-2233. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 26. O’Callaghan Y, O’Brien N. Bioaccessibility, cellular uptake and transepithelial transport of α‐tocopherol and retinol from a range of supplemented foodstuffs assessed using the caco‐2 cell model. Int J Food Sci Tech. 2010;45: 1436-1442. 27. General Food Distribution, Module 11. Part2: Technical Notes. Food basket commodities [Internet]. 2011 [cited 2016 August 16]. Available online: http://www.unscn.org/layout/modules/htp/pdf/M11P2.pdf. 28. Garrett DA, Failla ML, Sarama RJ. Development of an in-vitro digestion method to assess carotenoid bioavailability from meals. J Agric Food Chem. 1999;47: 4301-4309. 29. Jin F, Frohman C, Thannhauser T, Welch R, Glahn R. Effects of ascorbic acid, phytic acid and tannic acid on iron bioavailability from reconstituted ferritin measured by an in-vitro digestion-Caco-2 cell model. Br J Nutr. 2009;101: 972-981. 30. Etcheverry P, Miller D, Glahn R. A low-molecular-weight factor in human milk whey promotes iron uptake by Caco-2 cells. J Nutr. 2004;134: 93-98. 31. Liu C, Glahn RP, Liu RH. Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in-vitro digestion. J Agric Food Chem. 2004;52: 4330-4337. 32. Gallardo-Guerrero L, Gandul-Rojas B, Mínguez-Mosquera MI. Digestive stability, micellarization, and uptake by Caco-2 human intestinal cell of chlorophyll derivatives from different preparations of pea (Pisum sativum L.). J Agric Food Chem. 2008;56: 8379-8386. 33. Failla ML, Chitchumronchokchai C, Ferruzzi MG, Goltz SR, Campbell WW. Unsaturated fatty acids promote bioaccessibility and basolateral secretion of carotenoids and α-tocopherol by Caco-2 cells. 2014;5: 1101-1112. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 34. Latunde-Dada GO, Li X, Parodi A, Edwards C, Ellis P, Sharp PA. Micromilling enhances iron bioaccessibility from wholegrain wheat. J Agric Food Chem. 2014;62: 11222-11227. 35. Cilla A, Perales S, Lagarda M, Barbera R, Farre R. Iron bioavailability in fortified fruit beverages using ferritin synthesis by Caco-2 cells. J Agric Food Chem. 2008;56: 8699-8703. 36. Lindshield BL, King JL, Wyss A, Goralczyk R, Lu CH, Ford NA, Erdman JW, Jr. Lycopene biodistribution is altered in 15,15'-carotenoid monooxygenase knockout mice. J Nutr. 2008;138: 2367-2371. 37. Deming DM, Teixeira SR, Erdman JW, Jr. All-trans beta-carotene appears to be more bioavailable than 9-cis or 13-cis beta-carotene in gerbils given single oral doses of each isomer. J Nutr. 2002;132: 2700-2708. 38. Tan SY, Yeung CK, Tako E, Glahn RP, Welch RM, Lei X, Miller DD. Iron bioavailability to piglets from red and white common beans (Phaseolus vulgaris). J Agric Food Chem. 2008;56: 5008-5014. 39. Tako E, Beebe SE, Reed S, Hart JJ, Glahn RP. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr J. 2014;13:28. 40. Delimont NM, Fiorentino NM, Opoku-Acheampong AB, Joseph MV, Guo Q, Alavi S, Lindshield BL. Newly formulated, protein qualityenhanced, extruded sorghum-, cowpea-, corn-, soya-, sugar- and oilcontaining fortified-blendedfoodslead to adequatevitamin A and iron outcomesandimprovedgrowthcompared withnon-extrudedCSB+in rats. J Nutr Sci 2017;6:e18. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 41. Proulx AK, Reddy MB. Iron bioavailability of hemoglobin from soy root nodules using a Caco-2 cell culture model. J Agric Food Chem. 2006;54: 1518- 42. Ghavidel RA and Prakash J. The impact of germination and dehulling on nutrients, antinutrients, in-vitro iron and calcium bioavailability and in-vitro starch and protein digestibility of some legume seeds. LWT-Food Sci and Tech. 2007;40: 1292-1299. 43. Lestienne I, Buisson M, Lullien-Pellerin V, Picq C, Trèche S. Losses of nutrients and anti-nutritional factors during abrasive decortication of two pearl millet cultivars (Pennisetum glaucum). Food Chem. 2007;100: 1316-1323. 44. Gahlawat P, and Sehgal S. In-vitro starch and protein digestibility and iron availability in weaning foods as affected by processing methods. Plant Foods Hum Nutr. 1994;45: 165-173. 45. Chang S, Huang Z, Ma Y, Piao J, Yang X, Zeder C, Hurrell RF, Egli I. Mixture of ferric sodium ethylenediaminetetraacetate (NaFeEDTA) and ferrous sulfate: an effective iron fortificant for complementary foods for young Chinese children. Food Nutr Bull. 2012;33: 111-116. 46. Gibson RS, Bailey KB, Gibbs M, Ferguson EL. A review of phytate, iron, zinc, and calcium concentrations in plant-based complementary foods used in low-income countries and implications for bioavailability. Food Nutr Bull. 2010;31: S134-146. 47. Bothwell TH, and MacPhail AP. The potential role of NaFeEDTA as an iron fortificant. Int J Vitam Nutr Res. 2004;74: 421-434. 48. Failla, ML, Huo T, Thakkar, SK. In vitro screening of relative bioaccessibility of carotenoids from foods. Asia Pac. J. Clin. Nutr. 2008; 17(Suppl 1): 200-203. 49. Ariza-Nieto M, Blair MW, Welch RM, Glahn RP. Screening of iron bioavailability patterns in eight bean (Phaseolus vulgaris L.) genotypes using the Caco-2 cell in-vitro model. J Agric Food Chem. 2007;55: 7950-7956. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 50. Jovaní M, Barberá R, Farré R, Martín de Aguilera E. Calcium, iron, and zinc uptake from digests of infant formulas by Caco-2 cells. J Agric Food Chem. 2001;49: 3480-3485. 51. US Food and Drug Administration, Guidance for Industry: A Food Labeling Guide (15. Appendix G: Daily Values for Infants, Children Less Than 4 Years of Age, and Pregnant and Lactating Women. [Internet]. 2013 [updated 2013 January; cited 2018 January 4]. Available from: https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/LabelingNutrition/ucm2006828.htm. Table 1. Extruded fortified blended foods composition Sorghum-Cowpea Sorghum-Soy Corn-Soy Ingredient (%) blend blend blend (SCB, n=7) (SSB, n=3)* (CSB14, n=1) Sorghum flour 24.7 47.6 - Cowpea flour 38.6 - - Corn flour - - 48.1 Soy flour - 15.7 15.2 Sugar 15.0 15.0 15.0 Whey protein concentrate (WPC 80) 9.5 9.5 9.5 Vegetable oil 9.0 9.0 9.0 Vitamin and mineral premix 3.2 3.2 3.1 * For extruded SSB (full-fat soy), WPC80 was increased from 9.5% to 13% and vegetable oil was decreased from 9% to 5.5%. SCB; sorghum-cowpea blend, SSB; sorghum-soy blend, CSB; corn-soy blend. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 2. Non-extruded fortified blended foods and Cerelac compositions Non-Extruded FBFs Cerelac Corn-Soy Corn-Soy Ingredient (%) blend 13 Ingredient blend plus (CSB+) (CSB13) Cornmeal 69.5 - Wheat flour Soy flour, defatted 21.9 - Fat free milk Soybean oil, refined 5.5 - Sugar Minerals 3.0 - Milk fat Vitamin antioxidant premix 0.1 - Corn oil Corn (white or yellow) - 78.5 Palm oil Whole soybeans - 20.0 Calcium carbonate Vitamin/mineral - 0.2 Sodium phosphate Tri-calcium phosphate - 1.2 Bifidus cultures Potassium chloride - 0.2 Vitamin/ mineral For Cerelac, ingredient % was not available, ingredients were listed in the order on the label. FBF; Fortified blended foods, CSB; corn-soy blend. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 3. Fortified blended food and Cerelac macronutrient and micronutrient compositions (100 g) Extruded FBFs Non-extruded FBFs Nutrient Unit SCB/SSB/CSB14 CSB13 CSB+ Cerelac Energy kcal 380 386 380 400 Protein g 17 16 14 13 Fat g 8 9 6 10 †† Vitamin A mg 0.49 0.82 1.04 0.34 Thiamin (B ) mg 0.65 0.61 0.20 0.28 Riboflavin (B ) mg 0.93 0.48 1.4 0.8 Niacin (B ) mg 9.1 6.3 8.0 2.4 Pantothenic Acid (B ) mg 3.7 3.2 1.6 1.3 Vitamin B mg 0.8 0.5 1.0 0.3 Folic Acid (B ) mg 0.09 0.25 0.11 0.05 Vitamin B mg 0.002 0.001 0.002 0.001 Vitamin D mg 0.03 0.005 0.01 0.004 Vitamin E mg 13.2 1.0 8.3 3.0 Vitamin K mg 0.03 0.001 0.03 0.03 Vitamin C mg 40.0 40.0 90.0 16.0 Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Calcium mg 279.1 650.0 452.0 427.0 Total Iron (Fe) mg 13.0 10.6 6.5 5.3 Fe (FeSO ) mg 11.0 0.0 4.0 0.0 Fe (NaFeEDTA) mg 2.0 0.0 2.5 0.0 Fe (Ferrous Fumarate) mg 0.0 10.6 4.0 5.3 Iodine mg 0.23 0.0 0.04 0.03 Phosphorus mg 291.0 522.0 290.0 427.0 Potassium mg 163.2 563.0 140.0 NA Zinc mg 5.5 5.9 5.0 2.1 For Cerelac, macronutrient and micronutrient values were calculated based on the % Daily Value (% DV) information provided on the label, and daily values for children under 4-years provided by reference [51]. †† Extruded and non-extruded FBFs contained vitamin A in the form of retinyl palmitate, while Cerelac contained retinyl acetate. SCB; sorghum-cowpea blend, SSB; sorghum-soy blend, CSB; corn-soy blend. NA- not available. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 31 Table 4.Dry fortified blended food, Cerelac and aqueous fraction iron concentrations (mean ± SEM) Aqueous Dry FBF No Cereal Cereal type Legume fraction (mg/100 g) (µg/10 ml) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 19.5 0.93 ± 0.02 2 White Sorghum 1 Decorticated Cowpea 18.6 1.03 ± 0.06 ac 3 White Sorghum 1 Decorticated-C Cowpea 15.1 0.66 ± 0.11 ac 4 White Sorghum 2 Whole Cowpea 19.5 0.68 ± 0.17 ac 5 White Sorghum 2 Decorticated Cowpea 15.9 0.82 ± 0.03 ac 6 Red Sorghum Whole Cowpea 19.1 0.80 ± 0.08 ac 7 Red Sorghum Decorticated Cowpea 15.3 0.62 ± 0.18 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 23.4 0.95 ± 0.06 ac 9 White Sorghum 1 Decorticated Medium-Fat Soy 15.6 0.85 ± 0.05 10 White Sorghum 1 Whole Full-Fat Soy 20.7 0.92 ± 0.03 Corn-Soy blends ac 11 CSB14 Degermed corn-C Medium-Fat Soy 15.6 0.75 ± 0.09 12 CSB13 Cornmeal Defatted Soy Flour 31.8 0.17 ± 0.03 13 CSB+ Whole Corn Whole Soy 8.0 0.14 ± 0.03 Cerelac bc 14 Wheat Wheat Flour NA 11.4 0.42 ± 0.01 Within a column, means without a common superscript are significantly different (p<0.05). Aqueous fractions (n=2) from two different in-vitro digestion experiments (4 replicates). White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. C; Commercial; CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 5. Caco-2 cell ferritin levels following aqueous fraction treatment (n=2, mean ± SEM) Ferritin No Cereal Cereal type Legume (ng/mg) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 6.21 ± 1.68 2 White Sorghum 1 Decorticated Cowpea 4.66 ± 0.05 3 White Sorghum 1 Decorticated-C Cowpea 4.74 ± 0.05 4 White Sorghum 2 Whole Cowpea 6.52 ± 1.83 5 White Sorghum 2 Decorticated Cowpea 5.47 ± 2.05 6 Red Sorghum Whole Cowpea 5.24 ± 1.35 7 Red Sorghum Decorticated Cowpea 4.13 ± 1.01 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 6.51 ± 2.17 9 White Sorghum 1 Decorticated Medium-Fat Soy 5.97 ± 3.06 10 White Sorghum 1 Whole Full-Fat Soy 4.10 ± 1.22 Corn-Soy blends ab 11 CSB14 Degermed corn-C Medium-Fat Soy 6.78 ± 0.64 12 CSB13 Cornmeal Defatted Soy Flour 4.72 ± 0.73 ab 13 CSB+ Whole Corn Whole Soy 7.39 ± 1.76 Cerelac 14 Wheat Wheat Flour NA 4.89± 0.53 Controls Basal salt solution (negative control) 4.75 ± 1.04 ab FeSO (0.1 µg Fe/well) (positive control) 15.87 ± 5.73 FeSO (0.2 µg Fe/well) (positive control) 29.65 ± 3.26 Within a column, means without a common superscript are significantly different (p<0.05). White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full- fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. C; Commercial, CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 6. Dry fortified blended food, Cerelac and aqueous fraction vitamin A concentrations (n=2, mean ± SEM) Aqueous Dry FBF No Cereal Cereal type Legume fraction (mg/100 g) (ng/ml) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 0.75 60.8 ± 10.0 2 White Sorghum 1 Decorticated Cowpea 0.70 69.6 ± 15.9 3 White Sorghum 1 Decorticated-C Cowpea 0.54 50.8 ± 5.0 4 White Sorghum 2 Whole Cowpea 0.71 52.7 ± 7.7 5 White Sorghum 2 Decorticated Cowpea 0.56 80.1 ± 15.4 6 Red Sorghum Whole Cowpea 0.76 77.6 ± 2.8 7 Red Sorghum Decorticated Cowpea 0.54 67.4 ± 27.7 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 0.72 48.0 ± 4.0 9 White Sorghum 1 Decorticated Medium-Fat Soy 0.59 44.8 ± 10.9 10 White Sorghum 1 Whole Full-Fat Soy 0.75 49.1 ± 0.8 Corn-Soy blends 11 CSB14 Degermed corn-C Medium-Fat Soy 0.56 45.9 ± 12.8 12 CSB13 Cornmeal Defatted Soy Flour 1.16 33.0 ± 0.4 13 CSB+ Whole Corn Whole Soy 1.67 41.5 ± 28.2 Cerelac 14 Wheat Wheat Flour NA 0.30 55.6 ± 25.6 White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. No statistically significant differences. C; Commercial, CSB; corn-soy blend, NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 7. Caco-2 cell vitamin A concentrations following aqueous fraction treatment (n=2, mean ± SEM) Vitamin A No Cereal Cereal type Legume (µg/g cells) Sorghum-Cowpea blends 1 White Sorghum 1 Whole Cowpea 2.59 ± 0.35 2 White Sorghum 1 Decorticated Cowpea 2.52 ± 0.13 3 White Sorghum 1 Decorticated-C Cowpea 2.34 ± 0.15 4 White Sorghum 2 Whole Cowpea 2.23 ± 0.12 5 White Sorghum 2 Decorticated Cowpea 2.29 ± 0.15 6 Red Sorghum Whole Cowpea 1.89 ± 0.52 7 Red Sorghum Decorticated Cowpea 1.99 ± 0.14 Sorghum-Soy blends 8 White Sorghum 1 Whole Low-Fat Soy 1.96 ± 0.11 9 White Sorghum 1 Decorticated Medium-Fat Soy 2.13 ± 0.01 10 White Sorghum 1 Whole Full-Fat Soy 2.09 ± 0.69 Corn-Soy blends 11 CSB14 Degermed corn-C Medium-Fat Soy 1.96 ± 0.01 †† 12 CSB13 Cornmeal Defatted Soy Flour 2.03 †† 13 CSB+ Whole Corn Whole Soy 2.43 Cerelac 14 Wheat Wheat Flour NA 2.24 ± 0.10 Control Basal salt solution (negative control) 2.09 ± 0.21 White decorticated sorghum and degermed corn flour were sourced commercially and expected to have coarse particle size. Fat percentage in low-fat soy (1.85%); medium-fat soy (6.94%) and full-fat soy (16.93%). No significant differences. CSB13 and CSB+ are non-extruded FBFs, all other blends are extruded FBFs. †† For CSB13 and CSB+ (n=1) was included due to sample loss. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018 C; Commercial; CSB; corn-soy blend; NA; not applicable. Downloaded from https://academic.oup.com/cdn/advance-article-abstract/doi/10.1093/cdn/nzy021/4995903 by Ed 'DeepDyve' Gillespie user on 17 July 2018

Journal

Current Developments in NutritionOxford University Press

Published: May 14, 2018

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